Multivalent Immunoglobulin-Based Bioactive Assemblies

ABSTRACT

The present invention concerns methods and compositions for stably tethered structures of defined compositions, which may have multiple functionalities and/or binding specificities. Preferred embodiments concern hexameric stably tethered structures comprising one or more IgG antibody fragments and which may be monospecific or bispecific. The disclosed methods and compositions provide a facile and general way to obtain stably tethered structures of virtually any functionality and/or binding specificity. The stably tethered structures may be administered to subjects for diagnostic and/or therapeutic use, for example for treatment of cancer or autoimmune disease. The stably tethered structures may bind to and/or be conjugated to a variety of known effectors, such as drugs, enzymes, radionuclides, therapeutic agents and/or diagnostic agents.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/396,605 filed Mar. 3, 2009, which is a divisional of U.S. patentapplication Ser. No. 11/633,729 (now issued U.S. Pat. No. 7,527,787),filed Dec. 5, 2006, which is a continuation-in-part ofPCT/US2006/010762, filed Mar. 24, 2006; PCT/US2006/012084, filed Mar.29, 2006; PCT/US2006/025499, filed Jun. 29, 2006; U.S. patentapplication Ser. No. 11/389,358 (now issued U.S. Pat. No. 7,550,143),filed Mar. 24, 2006; Ser. No. 11/391,584 (now issued U.S. Pat. No.7,521,056), filed Mar. 28, 2006 and Ser. No. 11/478,021 (now issued U.S.Pat. No. 7,534,866), filed Jun. 29, 2006; which claimed priority toprovisional U.S. patent application Nos. 60/782,332, filed Mar. 14,2005; 60/728,292, filed Oct. 19, 2005, and 60/751,196, filed Dec. 16,2005. This application claims the benefit under 35 U.S.C. §119(e) toprovisional U.S. patent application No. 60/751,196, filed Dec. 16, 2005,and No. 60/864,530, filed Nov. 6, 2006. The text of each of theapplications cited above is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

Existing technologies for the production of antibody-based agents havingmultiple functions or binding specificities suffer a number oflimitations. For agents generated by recombinant engineering, suchlimitations may include high manufacturing cost, low expression yields,instability in serum, instability in solution resulting in formation ofaggregates or dissociated subunits, undefined batch composition due tothe presence of multiple product forms, contaminating side-products,reduced functional activities or binding affinity/avidity attributed tosteric factors or altered conformations, etc. For agents generated byvarious methods of chemical cross-linking, high manufacturing cost andheterogeneity of the purified product are two major limitations.

In recent years there has been an increased interest in antibodies orother binding moieties that can bind to more than one antigenicdeterminant (also referred to as epitopes). Generally, naturallyoccurring antibodies and monoclonal antibodies have two antigen bindingsites that recognize the same epitope. In contrast, bifunctional orbispecific antibodies (hereafter, only the term bispecific antibodieswill be used throughout) are synthetically or genetically engineeredstructures that can bind to two distinct epitopes. Thus, the ability tobind to two different antigenic determinants resides in the samemolecular construct.

Bispecific antibodies are useful in a number of biomedical applications.For instance, a bispecific antibody with binding sites for a tumor cellsurface antigen and for a T-cell surface receptor can direct the lysisof specific tumor cells by T cells. Bispecific antibodies recognizinggliomas and the CD3 epitope on T cells have been successfully used intreating brain tumors in human patients (Nitta, et al. Lancet. 1990;355:368-371). More recently, a new class of bispecific antibodies termed“bispecific T-cell engagers” (BiTEs) was reported to overcome thelimitations of most tumor-targeting bispecific antibodies that involvethe recruitment of effector cells for biological activities (Kufer, etal. Trends in Biotechnol. 2004; 22: 238-244). BiTEs are recombinantbispecific single-chain antibodies composed of two distinct single-chainFc fragments (scFvs) directed against a surface antigen on target cellsand CD3 on T cells joined in tandem via a flexible polypeptide linker(Mack, et al., Proc Natl Acad Sci U.S.A. 1995; 92: 7021-7025). BiTEs areproduced in mammalian cells and in constrast to other CD3-directedbispecific antibodies are capable of efficiently redirecting humanperipheral T lymphocytes to kill target cells without any requirementfor pre- or costimulation of the effector T cells (Mack, et al. JImmonol. 1997; 158: 3965-3970; Loffler, et al. Blood. 2000; 95:2098-2103). BiTE concentrations as low as 10-100 pg/mL (˜0.1-2 pM) wereshown to be sufficient for achieving half-maximal target cell lysis invitro (Dreier, et al. Int J Cancer. 2002; 100: 690-697) and tumor growthcould be prevented with sub-microgram amounts in mouse models (Dreier,et al. J Immunol. 2003; 170: 4397-4404; Schlereth et al. Cancer Res.2005; 65: 2882-2889).

Numerous methods to produce bispecific antibodies are known. Methods forconstruction and use of bispecific and multi-specific antibodies aredisclosed, for example, in U.S. Patent Application Publication No.20050002945, filed Feb. 11, 2004, the entire text of which isincorporated herein by reference. Bispecific antibodies can be producedby the quadroma method, which involves the fusion of two differenthybridomas, each producing a monoclonal antibody recognizing a differentantigenic site (Milstein and Cuello, Nature, 1983; 305:537-540). Thefused hybridomas are capable of synthesizing two different heavy chainsand two different light chains, which can associate randomly to give aheterogeneous population of 10 different antibody structures of whichonly one of them, amounting to ⅛ of the total antibody molecules, willbe bispecific, and therefore must be further purified from the otherforms, which even if feasible will not be cost effective. Furthermore,fused hybridomas are often less stable cytogenically than the parenthybridomas, making the generation of a production cell line moreproblematic.

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies, so that the resulting hybrid conjugate will bindto two different targets (Staerz, et al. Nature. 1985; 314:628-631;Perez, et al. Nature. 1985; 316:354-356). Bispecific antibodiesgenerated by this approach are essentially heteroconjugates of two IgGmolecules, which diffuse slowly into tissues and are rapidly removedfrom the circulation. Bispecific antibodies can also be produced byreduction of each of two parental monoclonal antibodies to therespective half molecules, which are then mixed and allowed to reoxidizeto obtain the hybrid structure (Staerz and Bevan. Proc Natl Acad SciUSA. 1986; 83:1453-1457). An alternative approach involves chemicallycross-linking two or three separately purified Fab′ fragments usingappropriate linkers. For example, European Patent Application 0453082disclosed the application of a tri-maleimide compound to the productionof bi- or tri-specific antibody-like structures. A method for preparingtri- and tetra-valent monospecific antigen-binding proteins bycovalently linking three or four Fab fragments to each other via aconnecting structure is provided in U.S. Pat. No. 6,511,663. All thesechemical methods are undesirable for commercial development due to highmanufacturing cost, laborious production process, extensive purificationsteps, low yields (<20%), and heterogeneous products.

Other methods include improving the efficiency of generating hybridhybridomas by gene transfer of distinct selectable markers viaretrovirus-derived shuttle vectors into respective parental hybridomas,which are fused subsequently (DeMonte, et al. Proc Natl Acad Sci USA.1990, 87:2941-2945); or transfection of a hybridoma cell line withexpression plasmids containing the heavy and light chain genes of adifferent antibody. These methods also face the inevitable purificationproblems discussed above.

A method to produce a recombinant bispecific antibody composed of Fabfragments from the same or different antibodies that are brought intoassociation by complementary interactive domains inserted into a regionof the antibody heavy chain constant region, was disclosed in U.S. Pat.No. 5,582,996. The complementary interactive domains are selected fromreciprocal leucine zippers or a pair of peptide segments, one containinga series of positively charged amino acid residues and the othercontaining a series of negatively charged amino acid residues. Onelimitation of such a method is that the individual Fab subunitscontaining the fused complementary interactive domains appear to havemuch reduced affinity for their target antigens unless both subunits arecombined.

Discrete V_(H) and V_(L) domains of antibodies produced by recombinantDNA technology may pair with each other to form a dimer (recombinant Fvfragment) with binding capability (U.S. Pat. No. 4,642,334). However,such non-covalently associated molecules are not sufficiently stableunder physiological conditions to have any practical use. Cognate V_(H)and V_(L) domains can be joined with a peptide linker of appropriatecomposition and length (usually consisting of more than 12 amino acidresidues) to form a single-chain Fv (scFv) with binding activity.Methods of manufacturing scFvs are disclosed in U.S. Pat. No. 4,946,778and U.S. Pat. No. 5,132,405. Reduction of the peptide linker length toless than 12 amino acid residues prevents pairing of V_(H) and V_(L)domains on the same chain and forces pairing of V_(H) and V_(L) domainswith complementary domains on other chains, resulting in the formationof functional multimers. Polypeptide chains of V_(H) and V_(L) domainsthat are joined with linkers between 3 and 12 amino acid residues formpredominantly dimers (termed diabodies). With linkers between 0 and 2amino acid residues, trimers (termed triabody) and tetramers (termedtetrabody) are favored, but the exact patterns of oligomerization appearto depend on the composition as well as the orientation of V-domains(V_(H)-linker-V_(L) or V_(L)-linker-V_(H)), in addition to the linkerlength.

Monospecific diabodies, triabodies, and tetrabodies with multiplevalencies have been obtained using peptide linkers consisting of 5 aminoacid residues or less. Bispecific diabodies, which are heterodimers oftwo different scFvs, each scFv consisting of the V_(H) domain from oneantibody connected by a short peptide linker to the V_(L) domain ofanother antibody, have also been made using a dicistronic expressionvector that contains in one cistron a recombinant gene constructcomprising V_(H1)-linker-V_(L2) and in the other cistron a secondrecombinant gene construct comprising V_(H2)-linker-V_(L1) (Holliger, etal. Proc Natl Acad Sci USA. 1993; 90: 6444-6448; Atwell, et al. MolImmunol. 1996; 33:1301-1302; Holliger, et al. Nature Biotechnol. 1997;15: 632-631; Helfrich, et al. Int. J Cancer. 1998; 76: 232-239;Kipriyanov, et al. Int J Cancer. 1998; 77: 763-772; Holliger, et al.Cancer Res. 1999; 59: 2909-2916).

More recently, a tetravalent tandem diabody (termed tandab) with dualspecificity has also been reported (Cochlovius, et al. Cancer Res. 2000;60: 4336-4341). The bispecific tandab is a dimer of two identicalpolypeptides, each containing four variable domains of two differentantibodies (V_(H1), V_(L1), V_(H2), V_(L2)) linked in an orientation tofacilitate the formation of two potential binding sites for each of thetwo different specificities upon self-association.

To date, the construction of a vector that expresses bispecific ortrispecific triabodies has not been achieved. However, polypeptidescomprising a collectin neck region are reported to trimerize (Hoppe, etal. FEBS Letters. 1994; 344: 191-195). The production of homotrimers orheterotrimers from fusion proteins containing a neck region of acollectin is disclosed in U.S. Pat. No. 6,190,886.

Methods of manufacturing scFv-based agents of multivalency andmultispecificity by varying the linker length were disclosed in U.S.Pat. No. 5,844,094, U.S. Pat. No. 5,837,242, and WO 98/44001. Methods ofmanufacturing scFv-based agents of multivalency and multispecificity byconstructing two polypeptide chains, one comprising of the V_(H) domainsfrom at least two antibodies and the other the corresponding V_(L)domains were disclosed in U.S. Pat. No. 5,989,830 and U.S. Pat. No.6,239,259. Common problems that have been frequently associated withgenerating scFv-based agents of multivalency and multispecificity byprior art methods are low expression levels, heterogenous product forms,instability in solution leading to aggregates, instability in serum, andimpaired affinity.

A recombinantly produced bispecific or trispecific antibody in which thec-termini of CH1 and C_(L) of a Fab are each fused to a scFv derivedfrom the same or different monoclonal antibodies was disclosed in U.S.Pat. No. 6,809,185. Major deficiencies of this “Tribody” technologyinclude impaired binding affinity of the appended scFvs, heterogeneityof product forms, and instability in solution leading to aggregates.

Thus, there remains a need in the art for a method of making multivalentstructures of either monospecificity or multiple specificities orfunctionalities, which are of defined composition, homogeneous purity,and unaltered affinity, and can be produced in high yields without therequirement of extensive purification steps. Furthermore, suchstructures must also be sufficiently stable in serum to allow in vivoapplications. A need exists for stable, multivalent structures ofmonospecificity or multiple specificities or functionalities that areeasy to construct and/or obtain in relatively purified form.

SUMMARY OF THE INVENTION

The present invention discloses a platform technology for generatingstably tethered structures that may be monospecific and/ormonofunctional, or may have multiple functions or binding specificities,and are suitable for in vitro as well as in vivo applications. Inpreferred embodiments, such stably tethered structures are produced ascomplexes of two components, referred herein as A and B, via specificinteractions between two distinct peptide sequences, one termeddimerization and docking domain (DDD) and the other anchoring domain(AD). In more preferred embodiments, the DDD sequences (shown for DDD1and DDD2 in FIG. 1) are derived from the regulatory (R) subunits of acAMP-dependent protein kinase (PKA), and the AD sequences (shown for AD1and AD2 in FIG. 2) are derived from a specific region found in variousA-kinase anchoring proteins (AKAPs) that mediates association with the Rsubunits of PKA. However, the skilled artisan will realize that otherdimerization and docking domains and anchoring domains are known and anysuch known domains may be used within the scope of the claimed subjectmatter. The disclosed methods and compositions enable site-directedcovalent or non-covalent association of any two complexes with theDDD/AD coupling system. The X-type four-helix bundle dimerization motifthat is a structural characteristic of the DDD (Newlon, et al. EMBO J.2001; 20: 1651-1662; Newlon, et al. Nature Struct Biol. 1999; 3:222-227) is found in other classes of proteins, such as the S100proteins (for example, S100B and calcyclin), and the hepatocyte nuclearfactor (HNF) family of transcriptional factors (for example, HNF-1a andHNF-1β). As S100 proteins have biological activities such astumorigenesis, they may be less desirable for such use.

Over 300 proteins that are involved in either signal transduction ortranscriptional activation contain a module of 65-70 amino acids termedthe sterile a motif (SAM) domain, which has a variation of the X-typefour-helix bundle present on its dimerization interface. For S100B, thisX-type four-helix bundle enables the binding of each dimer to two p53peptides derived from the c-terminal regulatory domain (residues367-388) with micromolar affinity (Rustandi, et al. Biochemistry. 1998;37: 1951-1960). Similarly, the N-terminal dimerization domain of HNF-1α(HNF-p1) was shown to associate with a dimer of DCoH (dimerizationcofactor for HNF-1) via a dimer of HNF-p1 (Rose, et al. Nature StructBiol. 2000; 7: 744-748). In alternative embodiments, these naturallyoccurring systems also may be utilized within the claimed methods andcompositions to provide stable multimeric structures with multiplefunctions or binding specificities. Other binding events such as thosebetween an enzyme and its substrate/inhibitor, for example, cutinase andphosphonates (Hodneland, et al. Proc Natl Acd Sci USA. 2002; 99:5048-5052), may also be utilized to generate the two associatingcomponents (the “docking” step), which are subsequently stabilizedcovalently (the “lock” step).

Other AD sequences of potential use may be found in Patent ApplicationSerial No. US20003/0232420A1, the entire text of which is incorporatedherein by reference.

In exemplary embodiments, one component of a binary complex, A, isproduced by linking a DDD sequence to the precursor of A, referred to asA, by recombinant engineering or chemical conjugation via a spacergroup, resulting in a structure of A/DDD, hereafter referred to as a. Asthe DDD sequence in a effects the spontaneous formation of a dimer, A isthus composed of a₂. The other component of a binary complex, B, isproduced by linking an AD sequence to the precursor of B, referred to asB, by recombinant engineering or chemical conjugation via a spacergroup, resulting in a structure of B/AD, hereafter referred to as b. Thefact that the dimeric structure contained in a₂ creates a docking sitefor binding to the AD sequence contained in b results in a readyassociation of a₂ and b to form a binary complex composed of a₂b. Invarious embodiments, this binding event is further stabilized with asubsequent reaction to covalently secure the two components of theassembly, for example via disulfide bridges, which occurs veryefficiently as the initial binding interactions orient the reactivethiol groups to ligate site-specifically.

By placing cysteine residues at strategic locations in both the DDD andAD sequences (as shown for DDD2 and AD2), the binding interactionbetween a₂ and b can be made covalent via disulfide bridges, therebyforming a stably tethered structure that renders in vivo applicationsmore feasible. The stably tethered structure also retains the fullfunctional properties of the two precursors A and B. The inventors areunaware of any prior art bispecific composition with this uniquecombination of features. The design disclosed above is modular innature, as each of the two precursors selected can be linked to eitherDDD or AD and combined afterwards. The two precursors can also be thesame (A=B) or different (A≠B). When A=B, the resulting a₂b complex iscomposed of a stably tethered assembly of three subunits, referred tohereafter as a₃. Materials that are amenable as precursors includeproteins, peptides, peptide mimetics, polynucleotides, RNAi,oligosaccharides, natural or synthetic polymeric substances,nanoparticles, quantum dots, and organic or inorganic compounds. Othernon-limiting examples of precursors of potential use are listed inTables 6-10 below.

In addition to the use of disulfide linkages for preventing thedissociation of the constituent subunits, other methods for enhancingthe overall stability of the stably tethered structure may be practiced.For example, various crosslinking agents or methods that arecommercially available or used in research may be selected for suchpurposes. A potentially useful agent is glutaraldehyde, which has beenwidely used for probing the structures of non-covalently associatedmultimeric proteins by cross-linking the constituent subunits to formstable conjugates (Silva, et al. Food Technol Biotechnol. 2004;42:51-56). Also of interest are two chemical methods involving oxidativecrosslinking of protein subunits. One is a proximity labeling techniquethat employs either hexahistidine-tagged proteins (Fancy, et al. ChemBiol. 1996; 3:551-559) or N-terminal glycine-glycine-histidine-taggedproteins (Brown, et al. Biochemistry. 1998; 37:4397-4406). These tagsbind Ni(II) tightly and, when oxidized with a peracid, a Ni(III) speciesis produced that is capable of mediating a variety of oxidativereactions, including protein-protein crosslinking. Another technique,termed PICUP (photo-induced crosslinking of unmodified proteins) uses[Ru(II)(bipy)₃]²⁺, ammonium persulfate, and visible light to induceprotein-protein crosslinking (Fancy and Kodadek. Proc Natl Acad Sci USA.1999; 96:6020-6024). However, as discussed below, numerous methods forchemically cross-linking peptide, polypeptide, protein or othermacromolecular species are known in the art and any such known methodmay be used to covalently stabilize the binary a₂b complex.

In more preferred embodiments, disclosed in more detail in Examples23-35 below, hexameric complexes may be formed that are eithermonospecific or bispecific. Such complexes may be formed, for example,as disclosed in FIG. 10, FIG. 11, and FIG. 13 by attaching one AD2 toeach of the C- or N-terminal ends of IgG moieties, which may then bindto DDD2-conjugated Fab fragments or other DDD2-conjugated antibodies orantibody fragments, to form a hexameric complex. As discussed inExamples 23-35, such monospecific or bispecific hexameric complexes showhigher binding affinity and increased efficacy compared to the parentantibodies or fragments. Numerous monospecific or bispecific hexamericstably tethered structures are disclosed in Examples 23-35. However, theskilled artisan will realize that the examples are not limiting and avariety of antigen-binding or other functional moieties may beincorporated into the disclosed hexameric structures, discussed in partin Tables 6-10.

The skilled artisan will realize that where the above discussion refersto IgG or Fab fragments, other types of antibodies, antibody fragments,or non-antibody proteins as discussed in more detail below may besubstituted. The stably tethered structures may comprise variouscombinations of antigen-binding components and/or effector components.For example, a bispecific antibody reacting with both activated plateletand tissue plasminogen activator (tPA) would not only prevent furtherclot formation by inhibiting platelet aggregation but also coulddissolve existing clot by recruiting endogenous tPA to the plateletsurface (Neblock et al., Bioconjugate Chem. 1991, 3:126-31). A stablytethered structure comprising a multivalent antibody binding componentagainst an internalizing tumor associated antigen (such as CD74) linkedto a toxin (such as a ribonuclease) would be valuable for selectivedelivery of the toxin to destroy the target tumor cell. A stablytethered structure comprising a soluble component of the receptor forIL-4R (sIL-4R) and a soluble component of the receptor for IL-13(sIL-13R) would be a potential therapeutic agent for treating asthma orallergy. A hexameric, monospecific stably tethered structure composed ofanti-GPIIb/IIIa Fab fragments should be more effective in preventingclot reformation than either the monovalent (ReoPro, Centocor) orbivalent analogs due to higher binding avidity. A stably tetheredstructure comprising multiple copies of a soluble component of TNFα-Rshould be more efficacious for arresting TNF than Enbrel (Amgen) in thetreatment of rheumatoid arthritis and certain other autoimmune diseases(AID).

The claimed methods and compositions also include conjugates composed ofone or more effectors or carriers linked to a stably tethered structure.The effectors or carriers may be linked to the stably tethered structureeither non-covalently or covalently, for example by chemicalcross-linking or by binding to a bispecific or multispecific stablytethered structure, with a first specificity for a disease-associatedtarget and a second specificity for an effector and/or hapten linked tothe effector(s), as discussed further below. Depending on the intendedapplications, the effector may be selected from a diagnostic agent, atherapeutic agent, a chemotherapeutic agent, a radioisotope, an imagingagent, an anti-angiogenic agent, a cytokine, a chemokine, a growthfactor, a drug, a prodrug, an enzyme, a binding molecule, a ligand for acell surface receptor, a chelator, an immunomodulator, anoligonucleotide, a hormone, a photodetectable label, a dye, a peptide, atoxin, a contrast agent, a paramagnetic label, an ultrasound label, apro-apoptotic agent, a liposome, a nanoparticle or a combinationthereof. Moreover, a conjugate may contain more than one effector, whichcan be the same or different, or more than one carrier, which can be thesame or different. Effectors and carriers can also be present in thesame conjugate. When the effector is a chelator, the resulting conjugateis usually further complexed with a metal, which can be eitherradioactive or non-radioactive. Conjugates containing carriers are alsofurther incorporated with agents of diagnostic or therapeutic functionsfor the intended applications.

In certain embodiments, the effectors or carriers may be administered toa subject after a stably tethered structure, for example inpre-targeting strategies discussed below. The stably tethered structuremay be first administered to the subject and allowed to localize in, forexample, a diseased tissue such as a tumor. The effectors or carriersmay be added subsequently and allowed to bind to the localized stablytethered structure. Where the effector or carrier is conjugated to atoxic moiety, such as a radionuclide, this pretargeting method reducesthe systemic exposure of the subject to toxicity, allowing aproportionately greater delivery of toxic agent to the targeted tissue.Optionally, a clearing agent may be administered to clear non-localizedstably tethered structures from circulation before administration of thetargetable construct. These methods are known in the art and describedin detail in U.S. Pat. No. 4,624,846, WO 92/19273, and Sharkey et al.,Int. J. Cancer 51: 266 (1992). An exemplary targetable construct mayhave a structure of X-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Y)-NH₂, where thecompound includes a hard acid cation chelator at X or Y, and a soft acidcation chelator at remaining X or Y; and wherein the compound furthercomprises at least one diagnostic or therapeutic cation, and/or one ormore chelated or chemically bound therapeutic agents, diagnostic agents,or enzymes. The diagnostic agent could be, for example, Gd(III),Eu(III), Dy(III), Pr(III), Pa(IV), Mn(II), Cr(III), Co(III), Fe(III),Cu(II), Ni(II), Ti(III), V(IV) ions or a radical. A second exemplaryconstruct may be of the formulaX-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-Lys(Y)-NH₂, where the compound includes ahard acid cation chelator or a soft acid chelator at X or Y, and nothingat the remaining X or Y; and wherein the compound further comprises atleast one diagnostic or therapeutic cation, and/or one or more chelatedor chemically bound therapeutic agents, diagnostic agents, or enzymes.In such embodiments, the A subunit may, for example, contain bindingsites for tumor associated antigens while the B subunit may contain abinding site for an effector or carrier or a hapten conjugated to aneffector or carrier.

The stably tethered structures of the present invention, including theirconjugates, are suitable for use in a wide variety of therapeutic anddiagnostic applications. For example, the hexavalent constructs based onantibody binding domains can be used for therapy where such a constructis not conjugated to an additional functional agent, in the same manneras therapy using a naked antibody. Alternatively, these stably tetheredstructures can be derivatized with one or more functional agents toenable diagnostic or therapeutic applications. The additional agent maybe covalently linked to the stably tethered structures usingconventional conjugation chemistries.

Methods of use of stably tethered structures may include detection,diagnosis and/or treatment of a disease or other medical condition. Suchconditions may include, but are not limited to, cancer, cardiovasculardisease, atherosclerosis, stroke, neurodegenerative disease, Alzheimer'sdisease, metabolic diseases, hyperplasia, diabetic retinopathy, maculardegeneration, inflammatory bowel disease, Crohn's disease, ulcerativecolitis, rheumatoid arthritis, sarcoidosis, asthma, edema, pulmonaryhypertension, psoriasis, corneal graft rejection, neovascular glaucoma,Osler-Webber Syndrome, myocardial angiogenesis, plaqueneovascularization, restenosis, neointima formation after vasculartrauma, telangiectasia, hemophiliac joints, angiofibroma, fibrosisassociated with chronic inflammation, lung fibrosis, amyloidosis, organtransplant rejection, deep venous thrombosis or wound granulation.

In particular embodiments, the disclosed methods and compositions may beof use to treat autoimmune disease, such as acute idiopathicthrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura,dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupuserythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, juvenile diabetes mellitus,Henoch-Schonlein purpura, post-streptococcalnephritis, erythemanodosurn, Takayasu's arteritis, Addison's disease, rheumatoid arthritis,multiple sclerosis, sarcoidosis, ulcerative colitis, erythemamultiforme, IgA nephropathy, polyarteritis nodosa, ankylosingspondylitis, Goodpasture's syndrome, thromboangitisubiterans, Sjogren'ssyndrome, primary biliary cirrhosis, Hashimoto's thyroiditis,thyrotoxicosis, scleroderma, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, perniciousanemia, rapidly progressive glomerulonephritis, psoriasis or fibrosingalveolitis.

Various embodiments may concern methods of treating inflammatory andimmune-dysregulatory diseases, infectious diseases, pathologicangiogenesis or cancer. In this application the stably tetheredstructures bind to two different targets selected from the groupconsisting of (A) proinflammatory effectors of the innate immune system,(B) coagulation factors, (C) complement factors and complementregulatory proteins, and (D) targets specifically associated with aninflammatory or immune-dysregulatory disorder or with a pathologicangiogenesis or cancer, wherein the latter target is not (A), (B), or(C). At least one of the targets is (A), (B) or (C). Suitablecombinations of targets are described in U.S. patent application Ser.No. 11/296,432, filed Dec. 8, 2005, entitled “Methods and Compositionsfor Immunotherapy and Detection of Inflammatory and Immune-DysregulatoryDisease, Infectious Disease, Pathologic Angiogenesis and Cancer,” thecontents of which are incorporated herein by reference in theirentirety. The proinflammatory effector of the innate immune system towhich the binding molecules may bind may be a proinflammatory effectorcytokine, a proinflammatory effector chemokine or a proinflammatoryeffector receptor. Suitable proinflammatory effector cytokines includeMIF, HMGB-1 (high mobility group box protein 1), TNF-α, IL-1, IL-4,IL-5, IL-6, IL-8, IL-12, IL-15, and IL-18. Examples of proinflammatoryeffector chemokines include CCL19, CCL21, IL-8, MCP-1, RANTES, MIP-1A,MIP-1B, ENA-78, MCP-1, IP-10, GROB, and Eotaxin. Proinflammatoryeffector receptors include IL-4R (interleukin-4 receptor), IL-6R(interleukin-6 receptor), IL-13R (interleukin-13 receptor), IL-15R(interleukin-15 receptor) and IL-18R (interleukin-18 receptor).

The binding molecule also may react specifically with at least onecoagulation factor, particularly tissue factor (TF) or thrombin. Inother embodiments, the binding molecule reacts specifically with atleast one complement factor or complement regulatory protein. Inpreferred embodiments, the complement factor is selected from the groupconsisting of C3, C5, C3a, C3b, and C5a. When the binding moleculereacts specifically with a complement regulatory protein, the complementregulatory protein preferably is selected from the group consisting ofCD46, CD55, CD59 and mCRP.

In certain embodiments, the stably tethered structures may be of use fortherapeutic treatment of cancer. It is anticipated that any type oftumor and any type of tumor antigen may be targeted. Exemplary types oftumors that may be targeted include acute lymphoblastic leukemia, acutemyelogenous leukemia, biliary cancer, breast cancer, cervical cancer,chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectalcancer, endometrial cancer, esophageal, gastric, head and neck cancer,Hodgkin's lymphoma, lung cancer, medullary thyroid cancer, non-Hodgkin'slymphoma, multiple myeloma, renal cancer, ovarian cancer, pancreaticcancer, glioma, melanoma, liver cancer, prostate cancer, and urinarybladder cancer.

Tumor-associated antigens that may be targeted include, but are notlimited to, carbonic anhydrase IX, A3, antigen specific for A33antibody, BrE3-antigen, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20,CD21, CD22, CD23, CD25, CD30, CD45, CD74, CD79a, CD80, HLA-DR, NCA95,NCA90, HCG and its subunits, CEA (CEACAM-5), CEACAM-6, CSAp, EGFR,EGP-1, EGP-2, Ep-CAM, Ba 733, HER2/neu, hypoxia inducible factor (HIF),KC4-antigen, KS-1-antigen, KS1-4, Le-Y, macrophage inhibition factor(MIF), MAGE, MUC1, MUC2, MUC3, MUC4, PAM-4-antigen, PSA, PSMA, RS5,S100, TAG-72, p53, tenascin, IL-6, IL-8, insulin growth factor-1(IGF-1), Tn antigen, Thomson-Friedenreich antigens, tumor necrosisantigens, VEGF, placenta growth factor (P1GF), 17-1A-antigen, anangiogenesis marker (e.g., ED-B fibronectin), an oncogene marker, anoncogene product, and other tumor-associated antigens. Recent reports ontumor associated antigens include Mizukami et al., (2005, Nature Med.11:992-97); Hatfield et al., (2005, Curr. Cancer Drug Targets 5:229-48);Vallbohmer et al. (2005, J. Clin. Oncol. 23:3536-44); and Ren et al.(2005, Ann. Surg. 242:55-63), each incorporated herein by reference.Particularly preferred embodiments may concern hexavalent, monospecificconstructs with binding sites for CD20 or CD22. Other preferredembodiments may concern a hexavalent bispecific construct with bindingsites for both CD20 and CD22.

Other embodiments may concern methods for treating a lymphoma, leukemia,or autoimmune disorder in a subject, by administering to the subject oneor more dosages of a stably tethered structure, where the binding siteof the second precursor bind to a lymphocyte antigen, and where thebinding site of the first precursor binds to the same or a differentlymphocyte antigen. The binding site or sites may bind a distinctepitope, or epitopes of an antigen selected from the group consisting ofCD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33,CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80, CD126, CD138,CD154, B7, MUC1, Ia, Ii, HM1.24, HLA-DR, tenascin, VEGF, P1GF, ED-Bfibronectin, an oncogene, an oncogene product, NCA 66a-d, necrosisantigens, IL-2, T101, TAG, IL-6, MIF, TRAIL-R1 (DR4) and TRAIL-R2 (DR5).The composition may be parenterally administered in a dosage of 20 to1500 milligrams protein per dose, 20 to 500 milligrams protein per dose,20 to 100 milligrams protein per dose, or 20 to 1500 milligrams proteinper dose, for example.

In other embodiments, the stably tethered structures may be of use totreat infection with pathogenic organisms, such as bacteria, viruses orfungi. Exemplary fungi that may be treated include Microsporum,Trichophyton, Epidermophyton, Sporothrix schenckii, Cryptococcusneoformans, Coccidioides immitis, Histoplasma capsulatum, Blastomycesdermatitidis or Candida albicans. Exemplary viruses include humanimmunodeficiency virus (HIV), herpes virus, cytomegalovirus, rabiesvirus, influenza virus, human papilloma virus, hepatitis B virus,hepatitis C virus, Sendai virus, feline leukemia virus, Reo virus, poliovirus, human serum parvo-like virus, simian virus 40, respiratorysyncytial virus, mouse mammary tumor virus, Varicella-Zoster virus,Dengue virus, rubella virus, measles virus, adenovirus, human T-cellleukemia viruses, Epstein-Barr virus, murine leukemia virus, mumpsvirus, vesicular stomatitis virus, Sindbis virus, lymphocyticchoriomeningitis virus or blue tongue virus. Exemplary bacteria includeBacillus anthracis, Streptococcus agalactiae, Legionella pneumophilia,Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae,Neisseria meningitidis, Pneumococcus spp., Hemophilis influenzae B,Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis or aMycoplasma.

Although not limiting, in various embodiments, the precursorsincorporated into the stably tethered structures may comprise one ormore proteins, such as a bacterial toxin, a plant toxin, ricin, abrin, aribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweedantiviral protein, gelonin, diphtherin toxin, Pseudomonas exotoxin,Pseudomonas endotoxin, Ranpirnase (Rap), Rap (N69Q), PE38, dgA, DT390,PLC, tPA, a cytokine, a growth factor, a soluble receptor component,surfactant protein D, IL-4, sIL-4R, sIL-13R, VEGF₁₂₁, TPO, EPO(erythropoietin), a clot-dissolving agent, an enzyme, a fluorescentprotein, sTNFα-R, an avimer, a scFv, a dsFv or a nanobody.

In other embodiments, an anti-angiogenic agent may form part or all of aprecursor or may be attached to a stably tethered structure. Exemplaryanti-angiogenic agents of use include angiostatin, baculostatin,canstatin, maspin, anti-VEGF antibodies or peptides, anti-placentalgrowth factor antibodies or peptides, anti-Flk-1 antibodies, anti-Flt-1antibodies or peptides, laminin peptides, fibronectin peptides,plasminogen activator inhibitors, tissue metalloproteinase inhibitors,interferons, interleukin 12, IP-10, Gro-β, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin 2,interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin,paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin,AGM-1470, platelet factor 4 or minocycline.

In still other embodiments, one or more therapeutic agents, such asaplidin, azaribine, anastrozole, azacytidine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin,10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin,irinotecan (CPT-11), SN-38, carboplatin, cladribine, cyclophosphamide,cytarabine, dacarbazine, docetaxel, dactinomycin, daunomycinglucuronide, daunorubicin, dexamethasone, diethylstilbestrol,doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholinodoxorubicin, doxorubicin glucuronide, epirubicin glucuronide, ethinylestradiol, estramustine, etoposide, etoposide glucuronide, etoposidephosphate, floxuridine (FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO),fludarabine, flutamide, fluorouracil, fluoxymesterone, gemcitabine,hydroxyprogesterone caproate, hydroxyurea, idarubicin, ifosfamide,L-asparaginase, leucovorin, lomustine, mechlorethamine,medroprogesterone acetate, megestrol acetate, melphalan, mercaptopurine,6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin,mitotane, phenyl butyrate, prednisone, procarbazine, paclitaxel,pentostatin, PSI-341, semustine streptozocin, tamoxifen, taxanes, taxol,testosterone propionate, thalidomide, thioguanine, thiotepa, teniposide,topotecan, uracil mustard, velcade, vinblastine, vinorelbine,vincristine, ricin, abrin, ribonuclease, onconase, rapLR1, DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtheria toxin, Pseudomonas exotoxin, Pseudomonas endotoxin, anantisense oligonucleotide, an interference RNA, or a combinationthereof, may be conjugated to or incorporated into a stably tetheredstructure.

Various embodiments may concern stably tethered structures and methodsof use of same that are of use to induce apoptosis of diseased cells.Further details may be found in U.S. Patent Application Publication No.20050079184, the entire text of which is incorporated herein byreference. Such structures may comprise precursors with binding affinityfor an antigen selected from the group consisting of CD2, CD3, CD8,CD10, CD21, CD23, CD24, CD25, CD30, CD33, CD37, CD38, CD40, CD48, CD52,CD55, CD59, CD70, CD74, CD80, CD86, CD138, CD147, HLA-DR, CEA, CSAp,CA-125, TAG-72, EFGR, HER2, HER3, HER4, IGF-1R, c-Met, PDGFR, MUC1,MUC2, MUC3, MUC4, TNFR1, TNFR2, NGFR, Fas (CD95), DR3, DR4, DR5, DR6,VEGF, P1GF, ED-B fibronectin, tenascin, PSMA, PSA, carbonic anhydraseIX, and IL-6. In more particular embodiments, a stably tetheredstructure of use to induce apoptosis may comprise monoclonal antibodies,Fab fragments, chimeric, humanized or human antibodies or fragments. Inpreferred embodiments, the stably tethered structure may comprisecombinations of anti-CD74×anti-CD20, anti-CD74×anti-CD22,anti-CD22×anti-CD20, anti-CD20×anti-HLA-DR, anti-CD19×anti-CD20,anti-CD20×anti-CD80, anti-CD2×anti-CD25, anti-CD8 X anti-CD25,anti-CD2×anti-CD147, anti-CEACAM5×anti-CD3, anti-CEACAM6×anti-CD3,anti-EGFR×anti-CD3, anti-HER2/neu×anti-CD3, anti-CD20×anti-CD3,anti-CD74×anti-CD3 and anti-CCD22×anti-CD3. In other preferredembodiments, the stably tethered structure may be a monospecific ormultispecific anti-CD20, anti-CD22, anti-HLA-DR and/or anti-CD74. Theskilled artisan will realize that a multivalent stably tetheredstructure may comprise multiple antigen-binding moieties that bind, forexample, to different epitopes of the CD20 or CD22 antigens, oralternatively may comprise multiple copies of a single antigen-bindingmoiety that all bind to the same epitope. In more preferred embodiments,the chimeric, humanized or human antibodies or antibody fragments may bederived from the variable domains of LL2 (anti-CD22), LL1 (anti-CD74),L243 (anti-HLA-DR) and A20 (anti-CD20).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows two exemplary DDD sequences. The underlined sequence inDDD1 (SEQ ID NO:1) corresponds to the first 44 amino-terminal residuesfound in the RIIα of human PKA. DDD2 (SEQ ID NO:2) differs from DDD1 inthe two amino acid residues at the N-terminus.

FIG. 2 shows two exemplary AD sequences. The underlined sequence of AD1(SEQ ID NO:3) corresponds to AKAP-is, which is an optimizedRII-selective peptide reported with a Kd of 0.4 nM. Also shown is AD2(SEQ ID NO:4).

FIG. 3 shows a schematic diagram of N-DDD2-Fab-hMN-14 (A), and theputative a₂ structure formed by DDD2-mediated dimerization (B).

FIG. 4 shows the design of the N-DDD2-VH-hMN-14-pdHL2 plasmid expressionvector.

FIG. 5 shows a schematic diagram of C-DDD2-Fab-hMN-14 (A), and theputative a₂ structure formed by DDD2-mediated dimerization (B).

FIG. 6 shows the design of the C-DDD2-VH-hMN-14-pdHL2 plasmid expressionvector.

FIG. 7 shows a schematic representation of (A) the noncovalent a₂bcomplex that is formed upon mixing N-DDD2-Fab-hMN-14 and h679-Fab-AD2under reducing conditions, and (B) the covalent TF1 structure formed bydisulfide bridges.

FIG. 8 shows a schematic diagram of TF2.

FIG. 9 is a sketch of C-H-AD2-IgG. (A) Arrangement of cDNA/polypeptidesequences for heavy chain-AD2 and light chain. (B) Schematicrepresentation of a C-H-AD2-IgG.

FIG. 10 is a schematic representation of a monospecific HIDS (hexavalentIgG-based DNL structure) resulting from the combination of C-H-AD2-IgGand Fab-DDD2 modules.

FIG. 11 is a schematic representation of a bispecific HIDS resultingfrom the combination of C-H-AD2-IgG and Fab-DDD2 modules.

FIG. 12 is a sketch of N-K-AD2-IgG. (A) Arrangement of cDNA/polypeptidesequences for heavy chain and AD2-light chain. (B) Schematicrepresentation of a N-K-AD2-IgG.

FIG. 13 is a schematic representation of a bispecific HIDS resultingfrom the combination of N-K-AD2-IgG and Fab-DDD2 modules.

FIG. 14 shows sketches of (A) Fc-AD2-pdHL2 shuttle vector, (B) IgG-pdHL2mammalian expression vector and (C) C-H-AD2-IgG-pdHL2 mammalianexpression vector.

FIG. 15 shows SE-HPLC analysis of Protein A-purified C-H-AD2-hLL2-IgG.Peaks representing monomeric and dimeric forms are indicated.

FIG. 16 shows SDS-PAGE analysis of Protein A-purified C-H-AD2-hLL2-IgGunder reducing and non-reducing conditions. Bands representing heavychain-AD2, heavy chain and kappa light chain are indicated for reducedlanes. Bands representing C-H-AD2-hLL2-IgG and the covalent dimer areindicated for non-reduced lanes. The positions of molecular weightmarkers are indicated.

FIG. 17 shows SE-HPLC analysis of Protein A-purified N-K-AD2-hLL2-IgG.(A) Peaks representing monomeric, dimeric and trimeric forms areindicated with arrows. (B) Analysis following reduction with glutathioneshowing that the dimeric and trimeric forms are converted to themonomeric form.

FIG. 18 shows sketches of postulated structures for (A) dimeric and (B)trimeric forms of N-K-AD2-hLL2-IgG, which are converted to (C) themonomeric form by mild reduction.

FIG. 19 shows SE-HPLC analysis of Protein A-purified Hex-hA20.

FIG. 20 shows SDS-PAGE analysis of six C-H-AD2-hLL2-IgG-based HIDS. (A)SDS-PAGE under non-reducing conditions. (B) SDS-PAGE under reducingconditions. Bands representing heavy chain-AD2, Fd-DDD2 and kappa lightchain are indicated by arrows. The positions of molecular weight markersare indicated.

FIG. 21 shows SE-HPLC analysis of Protein A-purified Hex-hLL2.

FIG. 22 shows SE-HPLC analysis of (A) DNL1 and (B) DNL1C.

FIG. 23 shows SE-HPLC analysis of DNL2.

FIG. 24 shows SDS-PAGE analysis of DNL3 and K-Hex-hA20 under reducingand non-reducing conditions. Bands representing heavy chain, AD2-kappachain, Fd-DDD2 and kappa light chain are shown in the reduced lanes.Bands representing DNL3 and K-Hex-hA20 are shown in the non-reducedlanes. The positions of molecular weight markers are indicated.

FIG. 25 shows SE-HPLC analysis of DNL3.

FIG. 26 shows the results of two competitive ELISA experiments tocompare the relative hA20/hLL2 binding avidities of DNL1, DNL2 Hex-hA20and Hex-hLL2 with the parental IgGs. Microtitre plates were coated withhA20 or hLL2 IgG at 5 μg/ml. Dilution series of the HIDS were mixed withanti-Ids specific to hA20 or hLL2 IgG, which was maintained at aconstant concentration (2 nM). The level of binding of the anti-Ids tothe coated wells was detected using peroxidase-conjugated-Goat anti-RatIgG and OPD substrate solution. The results are plotted as % inhibition(of anti-Id binding to coated wells) vs. concentration of HIDS. EC₅₀(the effective concentration resulting in 50% inhibition) values weredurived using Prism software. The HIDS were used to compete for bindingto (A) WI2 (hA20 Rat anti-Id) in hA20-coated wells or (B) WN (hLL2 Ratanti-Id) in hLL2-coated wells.

FIG. 27 shows the results of two competitive ELISA experiments tocompare the relative hA20/hLL2 binding avidities of DNL2 and DNL3.Experiments were carried out as described for FIG. 26. DNL2, DNL3 andthe parental IgGs were used to compete for binding to (A) WI2 (hA20 Ratanti-Id) in hA20-coated wells or (B) WN (hLL2 Rat anti-Id) inhLL2-coated wells.

FIG. 28 shows the result of cell counting assays following treatment ofDaudi lymphoma cells with DNL1, DNL2, Hex-hA20 or rituximab. Tissueculture flasks were inoculated with 1×10⁵ Daudi cells/ml in RPMI 1640media supplemented with one of the HIDS or rituximab at varyingconcentrations. Viable cells were counted daily using a Guava PCA. (A)Comparison growth curves following treatments at 10 nM concentrations.(B) Comparison of growth curves at selected concentrations.

FIG. 29 shows the results of a dose-response experiment for treatment ofDaudi cells with various HIDS. Cells were plated in 96-well plates at5,000 cells/well in RPMI 1640 media. Five-fold serial dilutions wereperformed in triplicate from concentrations of 2×10⁻⁸ down to 6.4×10⁻¹²M. The plates were incubated for four days, after which MTS reagent wasadded and the incubation was continued for an additional four hoursbefore reading the plates at 490 nm. The results are given as percent ofthe OD₄₉₀ for untreated wells vs. the log of the molar concentration ofHIDS. EC₄₀ (the effective concentration resulting in 40% growthinhibition) values were measured for each dose-response curve.

FIG. 30 shows the results of an in vivo therapy experiment where micebearing human Burkitt Lymphoma (Daudi) were treated with DNL2 orHex-hA20. Mice (4/group) were inoculated i.v. with 1.5×10⁷ Daudi cells(day 0). On days 1, 4 and 7, mice were administered either 4 μg or 20 μgof DNL2 or Hex-hA20 intraperitoneally (i.p.). Mice were sacrificed ifthey developed either hind-limb paralysis or lost >20% body weight. Theresults are plotted as % survival vs. time (days). Median survival andlong term survivors are shown.

FIG. 31 shows the relative dose-response curves generated using an MTSproliferation assay for Daudi cells, Raji cells and Ramos cells treatedwith a bispecific HID (DNL2-four hLL2 Fab fragments tethered to an hA20IgG) and a monospecific HID (Hex-hA20), compared with an hA20 IgGcontrol. In Daudi cells (top panel), DNL2 showed >100-fold and Hex-hA20showed >10,000 fold more potent antiproliferative activity than hA20IgG. In Raji cells (middle panel), Hex-hA20 displayed potentanti-proliferative activity, while DNL2 showed only minimal activity,compared to hA20 IgG. In Ramos cells (bottom panel), both DNLs andHex-hA20 displayed potent anti-proliferative activity compared to hA20IgG.

FIG. 32 shows the effects of cross-linking on the anti-proliferativeactivity of hA20 IgG, DNL2 and Hex-hA20. As shown in the Figure,cross-linking potentiated the anti-proliferative activity of hA20 IgG,but resulted in no enhancement of the activities of DNL2 or Hex-hA20.

FIG. 33 shows the stability of DNL1 and DNL2 in human serum, asdetermined using a bispecific ELISA assay. The protein structures wereincubated at 10 μg/ml in fresh pooled human sera at 37° C. and 5% CO₂for five days. For day 0 samples, aliquots were frozen in liquidnitrogen immediately after dilution in serum. ELISA plates were coatedwith an anti-Id to hA20 IgG and bispecific binding was detected with ananti-Id to hLL2 IgG. Both DNL1 and DNL2 were highly stable in serum andmaintained complete bispecific binding activity.

FIG. 34 illustrates the complement-dependent cytotoxicity (CDC) or lackthereof by DNL1, DNL2, Hex-hA20, hLL2, hA20-IgG and hA20-IgG-AD2.Surprisingly, although hA20 IgG and hA20-IgG-AD2 exhibited potent CDCactivity on Daudi cells in an in vitro assay, none of the hexavalent DNLstructures exhibited CDC activity in this assay. Both DNL2 and Hex-ha20comprise hA20-IgG-AD2, which showed CDC activity similar to hA20 IgG.

FIG. 35 shows the antibody-dependent cellular cytotoxicity (ADCC) ofDNL1, compared with hA20 IgG, Rituximab and hLL2 IgG, assayed withfreshly isolated peripheral blood mononuclear cells. Both rituximab andhA20 IgG had potent ADCC activity, while DNL1 did not exhibit anydetectable ADCC.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety.

In certain embodiments, novel stably tethered structures in the formatof a₂b and methods for making these complexes are provided. In general,the binary complexes are made up of a noncovalently linked homodimerstructure, referred to as A or a₂, with which a second structure,referred to as B or b, associates site-specifically. The resulting a₂bstructure may be stabilized by non-covalent, or preferably by covalentinteraction (e.g., disulfide bonds) between A and B. A is formed fromtwo identical subunits, where each subunit is composed of a precursorlinked to a peptide sequence, referred to as the dimerization anddocking domain (DDD), which in preferred embodiments is derived from acAMP-dependent protein kinase (PKA). The DDD domain contained in thesubunit associates spontaneously to form a stable homodimer, and thisassociation in turn produces a high affinity binding site for a peptidesequence, referred to as the anchoring domain (AD), which is found, forexample, in various A-kinase anchor proteins (AKAPs), and is containedin B. Thus, B is composed of a precursor linked to an AD.

Assembly of the binary complex occurs readily via interaction of the ADpeptide with the (DDD)₂ binding site. The DDD peptide may be insertedinto essentially any polypeptide sequence or tethered to any precursor,provided that such derivatization does not interfere with its ability todimerize, as well as to bind to the AD peptide. Likewise, the AD peptidemay be inserted into essentially any polypeptide sequence or tethered toany precursor provided that such derivatization does not interfere withits binding to the homodimer DDD binding site. This modular approach ishighly versatile and can be used to combine essentially any A with any Bto form a binary assembly that contains two subunits (a₂) derived fromthe precursor of A and one subunit (b) derived from the precursor of B.Where both precursors of A and B contain an antibody domain that canassociate with a second antibody domain to produce an antigen bindingsite (for example, a Fab or scFv), the resulting a₂b complex isbispecific and trivalent. In some embodiments, the binary complex may belinked, for example via chemical conjugation, to effectors, such asligands or drugs, to carriers, such as dextran or nanoparticles, or toboth effectors and carriers, to allow additional applications enabled bysuch modifications. In preferred embodiments, variations on this thememay be used to prepare hexameric complexes that are either homohexamersor heterohexamers.

As the stability of the binary complex depends primarily on the bindingaffinity of the DDD contained in A for the AD contained in B, which isestimated by equilibrium size-exclusion HPLC analysis to be no strongerthan 8 nM for two prototype a₂b structures (described in Example 5)formed between a C-terminally fused AD1 construct (h679-Fab-AD1,described in Example 3) to a C- or N-terminally fused DDD1 construct(C-DDD1-Fab-hMN-14 or N-DDD1-Fab-hMN-14, both described in Example 4),covalently linking A and B contained in the a₂b complex would preventundesirable dissociation of the individual subunits, therebyfacilitating in vivo applications. To stabilize the binary complex,cysteine residues may be introduced onto both the DDD and AD sequencesat strategic positions to enable the formation of disulfide linkagesbetween the DDD and AD. Other methods or strategies may be applied toeffect the formation of a stabilized complex via crosslinking a₂ and b.For example, the constituent subunits can be covalently linked to eachother in a less specific way with lower efficiency using glutaraldehydeor the PICUP method. Other known methods of covalent cross-linking mayalso be used.

DEFINITIONS

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein, the terms “and” and “or” may be used to mean either theconjunctive or disjunctive. That is, both terms should be understood asequivalent to “and/or” unless otherwise stated.

A “dimerization and docking domain (DDD)” refers to a peptide sequencethat allows the spontaneous dimer formation of two homomonomerscontaining the DDD sequence. The resulting homodimer contains a dockingsite within the DDD sequence for an anchoring domain. Although exemplaryDDD sequences may be obtained from cAMP-dependent protein kinase, otherknown DDD sequences may be utilized.

An “anchoring domain (AD)” is a peptide sequence that has bindingaffinity for a dimerized DDD sequence. Although exemplary AD sequencesmay be derived from any of the A-kinase anchor proteins (AKAPs), otherknown AD sequences may be utilized.

The term “precursor” is used according to its plain and ordinary meaningof a substance from which a more stable, definitive or end product isformed.

A “binding molecule,” “binding moiety” or “targeting molecule,” as usedherein, is any molecule that can specifically bind to a target molecule,cell, complex and/or tissue. A binding molecule may include, but is notlimited to, an antibody or a fragment, analog or mimic thereof, anavimer, an aptamer or a targeting peptide.

An “antibody,” as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion or analog of an immunoglobulin molecule, like an antibodyfragment.

An “antibody fragment” is a portion of an antibody such as F(ab)₂,F(ab′)₂, Fab, Fv, sFv and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody. The term “antibody fragment” also includes any synthetic orgenetically engineered protein that acts like an antibody by binding toa specific antigen to form a complex. For example, antibody fragmentsinclude isolated fragments consisting of the variable regions, such asthe “Fv” fragments consisting of the variable regions of the heavy andlight chains, recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”), and minimal recognition units (CDR) consisting of theamino acid residues that mimic the hypervariable region.

An “effector” is an atom, molecule, or compound that brings about achosen result. An effector may include a therapeutic agent and/or adiagnostic agent.

A “therapeutic agent” is an atom, molecule, or compound that is usefulin the treatment of a disease. Examples of therapeutic agents includeantibodies, antibody fragments, drugs, toxins, enzymes, nucleases,hormones, immunomodulators, antisense oligonucleotides, smallinterfering RNA (siRNA), chelators, boron compounds, photoactive agents,dyes, and radioisotopes. Other exemplary therapeutic agents and methodsof use are disclosed in U.S. Patent Publication Nos. 20050002945,20040018557, 20030148409 and 20050014207, each incorporated herein byreference.

A “diagnostic agent” is an atom, molecule, or compound that is useful indiagnosing a disease. Useful diagnostic agents include, but are notlimited to, radioisotopes, dyes (such as with the biotin-streptavidincomplex), contrast agents, fluorescent compounds or molecules, andenhancing agents (e.g., paramagnetic ions) for magnetic resonanceimaging (MRI).

An “immunoconjugate” is a conjugate of a binding molecule (e.g., anantibody component) with an atom, molecule, or a higher-orderedstructure (e.g., with a carrier, a therapeutic agent, or a diagnosticagent).

A “naked antibody” is an antibody that is not conjugated to any otheragent.

A “carrier” is an atom, molecule, or higher-ordered structure that iscapable of associating with a therapeutic or diagnostic agent tofacilitate delivery of such agent to a targeted cell. Carriers mayinclude lipids (e.g., amphiphilic lipids that are capable of forminghigher-ordered structures), polysaccharides (such as dextran), or otherhigher-ordered structures, such as micelles, liposomes, ornanoparticles.

As used herein, the term “antibody fusion protein” is a recombinantlyproduced antigen-binding molecule in which two or more of the same ordifferent scFv or antibody fragments with the same or differentspecificities are linked. Valency of the fusion protein indicates howmany binding arms or sites the fusion protein has to a single antigen orepitope; i.e., monovalent, bivalent, trivalent or multivalent. Themultivalency of the antibody fusion protein means that it can takeadvantage of multiple interactions in binding to an antigen, thusincreasing the avidity of binding to the antigen. Specificity indicateshow many antigens or epitopes an antibody fusion protein is able tobind; i.e., monospecific, bispecific, trispecific, multispecific. Usingthese definitions, a natural antibody, e.g., an IgG, is bivalent becauseit has two binding arms but is monospecific because it binds to oneepitope. Monospecific, multivalent fusion proteins have more than onebinding site for an epitope but only binds to one such epitope, forexample a diabody with two binding site reactive with the same antigen.The fusion protein may comprise a single antibody component, amultivalent or multispecific combination of different antibodycomponents, or multiple copies of the same antibody component. Thefusion protein may additionally comprise an antibody or an antibodyfragment and a therapeutic agent. Examples of therapeutic agentssuitable for such fusion proteins include immunomodulators(“antibody-immunomodulator fusion protein”) and toxins (“antibody-toxinfusion protein”). One preferred toxin comprises a ribonuclease (RNase),preferably a recombinant RNase.

An antibody or immunoconjugate preparation, or a composition describedherein, is said to be administered in a “therapeutically effectiveamount” if the amount administered is physiologically significant. Anagent is physiologically significant if its presence results in adetectable change in the physiology of a recipient mammal. In particularembodiments, an antibody preparation is physiologically significant ifits presence invokes an antitumor response or mitigates the signs andsymptoms of an autoimmune disease state. A physiologically significanteffect could also be the evocation of a humoral and/or cellular immuneresponse in the recipient mammal leading to growth inhibition or deathof target cells. A “therapeutically effective amount” is not limited tothe amount of an agent that produces the most preferred effect in asubject, but may refer to an amount that results in any of the possibleknown effects of the agent on a subject, cell, tissue or organ.

Methods to Generate a Stably Tethered Assembly of Modular Subunits

The disclosed methods and compositions provide a platform technology forgenerating a stably tethered assembly of modular subunits. Oneembodiment concerns a stably tethered binary complex formed from twodefined components, A and B, which are preferably produced separately.However, in alternative embodiments both A and B may be producedtogether, for example by transfecting a single cell line with a vectorthat codes for both A and B, or with two different vectors thatseparately encode A and B. Separate production is preferred where A andB are both Fab fragments, as otherwise co-production would result inheterogenous products due to light chain scrambling.

In some embodiments, A, consisting of two identical subunits (a₂), iscombined with B, consisting of one subunit (b), to form an assembly inthe configuration of a₂b. The association of A and B is site-specificand spontaneous, due to the strong binding interaction between the DDDand AD sequences that are built into A and B, respectively. Both A and Bcan be any entity and the precursor of A to which the DDD is linked maybe different from or the same as the precursor of B to which the AD islinked. In the latter case, the resulting a₂b complex, referred to asa₃, is composed of three subunits, each containing the same precursorbut linked to both DDD and AD.

The modular nature of the claimed methods and compositions allows thecombination of any A with any B. There is essentially no limit on thetypes of precursors that can be attached to or incorporated into A andB, so long as they do not interfere with the dimerization of DDD or thebinding of DDD to AD. When constructed by recombinant engineering, A andB can be produced independently in a different host cell, purified, andstored (or alternatively produced in the same host cell as discussedabove). However, the need for purification of A and B prior to assemblyis not absolutely required. Cell extracts or culture media containing Aand B may be mixed directly under appropriate conditions to effect theformation of the binary complex, which may then be stabilized bydisulfide linkages upon oxidation, and purified afterwards. In certainapplications, it may be desirable to conjugate B, after purification andbefore combining with A, with effectors or carriers. Alternatively, itmay be desirable to conjugate A, after purification and before combiningwith B, with effectors or carriers. It may also be desirable to modifyboth A and B with effectors or carriers before combining. In addition,conjugation of the a₂b complex with effectors or carriers may also bedesirable in certain applications. Where A and B are produced in thesame host cell, they may spontaneously assemble into an a₂b complex.

Preferred embodiments take advantage of the specific protein/proteininteractions between cAMP-dependent protein kinase (PKA) regulatorysubunits and A-kinase anchor proteins (AKAP) anchoring domains thatoccur in nature. PKA was first reported in 1968 (See Walsh et al., J.Biol. Chem. 243:3763-65 (1968)). The structure of the holoenzyme, whichconsists of two catalytic subunits that are held in an inactive form bya regulatory (R) subunit dimer, was elucidated in the mid 1970s (SeeCorbin et al., J. Biol. Chem. 248:1813-21 (1973)). Two types of Rsubunits (RI and RII) are found and each has α and β isoforms. The Rsubunits have been isolated only as stable dimers and the dimerizationdomain has been shown to consist of the first 44 amino-terminal residues(See Hausken et al., J. Biol. Chem. 271:29016-22 (1996)). The signalingspecificity of PKA, which is a broad-spectrum serine/threonine kinase,is achieved through compartmentalization of the holoenzyme via dockingproteins called A-kinase anchor proteins (AKAPs) (Scott et al., J. Biol.Chem. 265:21561-66 (1990)).

The first AKAP, microtubule-associated protein-2, was characterized in1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 81:6723-27 (1984)). Todate, more than 50 structurally diverse AKAPs have been identified inspecies ranging from yeast to humans (See Wong et al., Nat Rev Mol CellBiol. 12:959-70 (2004)). The PKA anchoring domain of AKAPs is anamphipathic helix of 14-18 residues (See Carr et al. J. Biol. Chem.266:14188-92 (1991)). The amino acid sequences of the PKA anchoringdomain are quite diverse among AKAPs and the binding affinities for RIIdimers ranges from 2-90 nM while the binding affinities for RI dimers isabout 100-fold weaker (See Alto et al. Proc. Natl. Acad. Sci USA.100:4445-50 (2003)). The anchoring domain binds to a hydrophobic surfaceon RII dimers formed by the first amino terminal 23 residues of RII(Colledge et al., Trends Cell Biol. 6:216-21 (1999)). Thus, the RIIdimerization domain and AKAP binding domain are both located within thesame 44 amino acid sequence. Further, AKAPs will only bind to RIIdimers, not monomers. A structural model of this interaction is shown inFIG. 7.

The non-covalent complexes formed via the interaction of the DDD and ADsequences may be covalently stabilized to allow in vivo applications.This may be achieved through the introduction of cysteine residues intoboth the DDD and AD sequences at strategic positions (as those shown forDDD2 and AD2) to facilitate the formation of disulfide linkages.Alternatively, other known types of covalent cross-linking may beemployed.

The two components of the binary complex (A and B), when produced byrecombinant engineering, may be synthesized within the same host cell,or more preferably in two separate host cell lines. An expression vectordirecting the synthesis of A will contain the DNA sequences of apolypeptide of interest (A) fused to a sequence encoding the DDD of aPKA R-subunit, such as DDD1 or DDD2, which may consist of the first 30or more amino acids of RIα, RIβ, RIIα, RIIβ, or any natural or syntheticfunctional analog. The DDD can be coupled to the amino-terminal orcarboxyl terminal end of A, either directly or preferably with a spacercontaining an appropriate length and composition of amino acid residues.Alternatively, the DDD can be positioned internally within the fusionprotein provided that the binding activity of the DDD and the desiredactivity of the polypeptide fusion partner are not compromised. Uponsynthesis, the A/DDD fusion protein will form exclusively a stablehomodimer with DDD1, or predominantly a stable homotetramer with DDD2.Methods for forming stable homohexamers or heterohexamers are discussedbelow in the Examples.

A second expression cassette directing the synthesis of B, which can bein the same vector that directs the synthesis of A or preferably anindependent one, will contain the DNA sequences of a polypeptide ofinterest (B) fused to a sequence encoding an anchoring domain (AD), suchas AD1 or AD2, which can be derived from any AKAP protein, or a naturalor synthetic analog as disclosed in US 2003/0232420A1, incorporatedherein by reference. The AD can be coupled to the amino-terminal orcarboxyl terminal end of B, either directly or preferably with a spacercontaining appropriate length or composition of amino acid residues.Mixing the B/AD2 fusion protein (b) with the A/DDD2 fusion protein(predominantly a₄) in the presence of a disulfide reducing agent resultsin a binary complex consisting of a₂b, which is subsequently stabilizedwith the formation of disulfide bonds facilitated by the addition of asuitable oxidizing agent such as dimethyl sulfoxide (DMSO).

The modular nature of the subunits allows the combination of anyDDD2-polypeptide dimer with any AD2-polypeptide. Stocks of a variety ofmodular subunits can be maintained individually either as purifiedproducts or unpurified cell culture supernatants and subsequentlycombined to obtain various a₂b structures when desired.

A further embodiment is that effectors, such as drugs or chelators, orcarriers, such as dextran or nanoparticles, may be coupled usingappropriate conjugation chemistry to either A or B following itspurification. Alternatively, such modifications can be made to thestructure after its formation and purification, or to both A and Bbefore mixing.

Stably Tethered Assembly of Modular Subunits Derived from RecombinantAntibody Binding Domains

The disclosed methods and compositions are useful for providingrecombinant antibody-based multivalent binding structures, which can bemonospecific or bispecific. For example, the DDD2 sequence can be fusedto a single chain Fv to obtain monospecific binding structures. Moregenerally, a DDD sequence can be fused to an antibody variable domainthat can associate with a complementary antibody variable domain to forman antigen-binding site. For example, the DDD1 or DDD2 sequence can befused to an antibody sequence containing a V_(H) domain and a CH1 domain(Fd/DDD), or alternatively to a V_(L) domain and a CL domain (L/DDD).The Fd/DDD or L/DDD can then associate with a complementary L or Fd,respectively, to form a Fab/DDD and further a dimer of Fab/DDD1 or atetramer/dimer of Fab/DDD2.

Similarly, an AD sequence like AD2 can be fused to a single chain Fv, ormore generally, to an antibody sequence containing a VH domain and a CH1domain (Fd/AD2), which forms a Fab/AD2 when paired with a cognateL-chain. Alternatively, an AD sequence like AD2 may be fused to anantibody sequence containing a VL domain and a CL domain, which forms aFab/AD2 when paired with a cognate Fd chain. Mixing a tetramer/dimer ofFab/DDD2 with Fab/AD2 followed by reduction and oxidation results in astably tethered assembly of a trivalent binding structure, which can bemonospecific or bispecific.

The V_(H) and V_(L) regions of the binding structure may be derived froma “humanized” monoclonal antibody or from a human antibody.Alternatively, the V_(H) and/or V_(L) regions may comprise a sequencederived from human antibody fragments isolated from a combinatorialimmunoglobulin library. See, for example, Barbas et al., METHODS: Acompanion to Methods in Enzymology 2: 119 (1991), and Winter et al.,Ann. Rev. Immunol. 12: 433 (1994). Cloning and expression vectors thatare useful for producing a human immunoglobulin phage library can beobtained, for example, from STRATAGENE Cloning Systems (La Jolla,Calif.).

The human antibody V_(H) or V_(L) sequence may be derived from a humanmonoclonal antibody produced in a mouse. Such antibodies are obtainedfrom transgenic mice that have been “engineered” to produce specifichuman antibodies in response to antigenic challenge. In this technique,elements of the human heavy and light chain loci are introduced intostrains of mice derived from embryonic stem cell lines that containtargeted disruptions of the endogenous heavy chain and light chain loci.The transgenic mice can synthesize human antibodies specific for humanantigens, and the mice can be used to produce human antibody-secretinghybridomas. Methods for obtaining human antibodies from transgenic miceare described by Green et al., Nature Genet. 7: 13 (1994), Lonberg etal., Nature 368: 856 (1994), and Taylor et al., Int. Immun. 6: 579(1994).

General Methods for the Production of Recombinant Fusion ProteinsContaining Antibody Fragments

Nucleic acid sequences encoding antibody fragments that recognizespecific epitopes can be obtained by techniques that are well known inthe art. For example, hybridomas secreting antibodies of a desiredspecificity can be used to obtain antibody-encoding DNA that can beprepared using known techniques, for example, by PCR or by traditionalcDNA cloning techniques. Alternatively, Fab′ expression libraries orantibody phage display libraries can be constructed to screen forantibody fragments having a desired specificity.

The nucleic acid encoding the antibody fragment can then be ligated,directly or via a sequence that encodes a peptide spacer, to nucleicacid encoding either the DDD or the AD. Methods of producing nucleicacid sequences encoding these types of fusion proteins are well known inthe art and are further provided in the Examples.

In another embodiment, additional amino acid residues may be added toeither the N- or C-terminus of the modular subunit composed of A/DDD orB/AD, where the exact fusion site may depend on whether the DDD or theAD are attached to the N- or C-terminus (or at an internal position).The additional amino acid residues may comprise a peptide tag, a signalpeptide, a cytokine, an enzyme (for example, a pro-drug activatingenzyme), a hormone, a toxin, a peptide drug, a membrane-interactingpeptide, or other functional proteins.

Methods for producing recombinant proteins in a desired host cell arewell known in the art. To facilitate purification, the stably tetheredstructures may contain suitable peptide tags, such as the FLAG sequenceor the poly-HIS sequence, to facilitate their purification with arelevant affinity column.

A exemplary expression system suitable for expressing the constituentsubunits of the stably tethered structures is the pdHL2 vector, whichhas an amplifiable murine dhfr gene that allows selection andamplification by methotrexate treatment. See Gillies et al., J. Immunol.Methods 125:191 (1989). The pdHL2 vector provides independent expressionof two genes that are separately controlled by two metallothioninepromoters and IgH enhancers.

Suitable host cells or cell lines for the expression of the constituentsubunits of the stably tethered structures of are known to one skilledin the art. The use of a human host cell would enable any expressedmolecules to be modified with human glycosylation patterns. However,there is no indication that a human host cell is essential or preferredfor the disclosed methods.

As an illustration, a murine myeloma cell line such as Sp2/0 can betransfected by electroporation with linearized pdHL2 vector that encodesa constituent subunit of the stably tethered structures. Selection canbe initiated 48 hours after transfection by incubating cells with mediumcontaining 0.05-0.1 μM methotrexate. The clones selected can then beamplified by a stepwise increase in methotrexate concentration up to 5μM.

Conjugates of the Stably Tethered Structures

Additional moieties can be conjugated to the stably tethered structuresdescribed above. For example, drugs, toxins, radioactive compounds,enzymes, hormones, cytotoxic proteins, chelates, cytokines, and otherfunctional agents may be conjugated to one or more subunits of thestably tethered structures. Conjugation can be via, for example,covalent attachments to amino acid residues containing amine, carboxyl,thiol or hydroxyl groups in the side-chains. Various conventionallinkers may be used for this purpose, for example, diisocyanates,diisothiocyanates, bis(hydroxysuccinimide) esters, carbodiimides,maleimide-hydroxysuccinimide esters, glutaraldehyde and the like.Conjugation of agents to the stably tethered structures preferably doesnot significantly affect the activity of each subunit contained in theunmodified structures. Conjugation can be carried out separately to themodular subunits and the subunits then allowed to assemble into thestably tethered construct, or alternatively conjugation may be carriedout using the fully formed stably tethered construct or any intermediatein the formation of the stably tethered construct. In addition,cytotoxic or other agents may be first coupled to a polymeric carrier,which is then conjugated to a stably tethered structure. For thismethod, see Ryser et al., Proc. Natl. Acad. Sci. USA, 75:3867-3870,1978; U.S. Pat. No. 4,699,784 and U.S. Pat. No. 4,046,722, which areincorporated herein by reference.

The conjugates described herein can be prepared by various methods knownin the art. For example, a stably tethered structure can be radiolabeledwith ¹³¹I and conjugated to a lipid, such that the resulting conjugatecan form a liposome. The liposome may incorporate one or moretherapeutic agents (e.g., a drug such as FUdR-dO) or diagnostic agents.Alternatively, in addition to the carrier, a stably tethered structuremay be conjugated to ¹³¹I (e.g., at a tyrosine residue) and a drug(e.g., at the epsilon amino group of a lysine residue), and the carriermay incorporate an additional therapeutic or diagnostic agent.Therapeutic and diagnostic agents may be covalently associated with oneor more than one subunit of the stably tethered structures.

The formation of liposomes and micelles is known in the art. See, e.g.,Wrobel and Collins, Biochimica et Biophysica Acta (1995), 1235: 296-304;Lundberg et al., J. Pharm. Pharmacol. (1999), 51:1099-1105; Lundberg etal., Int. J. Pharm. (2000), 205:101-108; Lundberg, J. Pharm. Sci.(1994), 83:72-75; Xu et al., Molec. Cancer Ther. (2002), 1:337-346;Torchilin et al., Proc. Nat'l. Acad. Sci., U.S.A. (2003), 100:6039-6044;U.S. Pat. No. 5,565,215; U.S. Pat. No. 6,379,698; and U.S. 2003/0082154.

Nanoparticles or nanocapsules formed from polymers, silica, or metals,which are useful for drug delivery or imaging, have been described aswell. See, e.g., West et al., Applications of Nanotechnology toBiotechnology (2000), 11:215-217; U.S. Pat. No. 5,620,708; U.S. Pat. No.5,702,727; and U.S. Pat. No. 6,530,944. The conjugation of antibodies orbinding molecules to liposomes to form a targeted carrier fortherapeutic or diagnostic agents has been described. See, e.g., Bendas,Biodrugs (2001), 15:215-224; Xu et al., Mol. Cancer Ther (2002),1:337-346; Torchilin et al., Proc. Nat'l. Acad. Sci. U.S.A (2003),100:6039-6044; Bally, et al., J. Liposome Res. (1998), 8:299-335;Lundberg, Int. J. Pharm. (1994), 109:73-81; Lundberg, J. Pharm.Pharmacol. (1997), 49:16-21; Lundberg, Anti-cancer Drug Design (1998),13: 453-461. See also U.S. Pat. No. 6,306,393; U.S. Ser. No. 10/350,096;U.S. Ser. No. 09/590,284, and U.S. Ser. No. 60/138,284, filed Jun. 9,1999. All these references are incorporated herein by reference.

A wide variety of diagnostic and therapeutic agents can beadvantageously used to form the conjugates of the stably tetheredstructures, or may be linked to haptens that bind to a recognition siteon the stably tethered structures. Diagnostic agents may includeradioisotopes, enhancing agents for use in MRI or contrast agents forultrasound imaging, and fluorescent compounds. Many appropriate imagingagents are known in the art, as are methods for their attachment toproteins or peptides (see, e.g., U.S. Pat. Nos. 5,021,236 and 4,472,509,both incorporated herein by reference). Certain attachment methodsinvolve the use of a metal chelate complex employing, for example, anorganic chelating agent such a DTPA attached to the protein or peptide(U.S. Pat. No. 4,472,509).

In order to load a stably tethered structure with radioactive metals orparamagnetic ions, it may be necessary to first react it with a carrierto which multiple copies of a chelating group for binding theradioactive metals or paramagnetic ions have been attached. Such acarrier can be a polylysine, polysaccharide, or a derivatized orderivatizable polymeric substance having pendant groups to which can bebound chelating groups such as, e.g., ethylenediaminetetraacetic acid(EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins,polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and thelike known to be useful for this purpose. Carriers containing chelatesmay be coupled to the stably tethered structure using standardchemistries in a way to minimize aggregation and loss ofimmunoreactivity.

Other, more unusual, methods and reagents that may be applied forpreparing such conjugates are disclosed in U.S. Pat. No. 4,824,659,which is incorporated by reference herein in its entirety. Particularlyuseful metal-chelate combinations include 2-benzyl-DTPA and itsmonomethyl and cyclohexyl analogs, used with diagnostic isotopes in thegeneral energy range of 60 to 4,000 keV. Some useful diagnostic nuclidesmay include ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ⁹⁴Tc,^(94m)Tc, ^(99m)Tc, or ¹¹¹In. The same chelates complexed withnon-radioactive metals, such as manganese, iron and gadolinium, areuseful for MRI, when used along with the stably tethered structures andcarriers described herein. Macrocyclic chelates such as NOTA, DOTA, andTETA are of use with a variety of metals and radiometals, mostparticularly with radionuclides of gallium, yttrium and copper,respectively. Such metal-chelate complexes can be made very stable bytailoring the ring size to the metal of interest. Other ring-typechelates, such as macrocyclic polyethers for complexing ²²³Ra, may beused.

Therapeutic agents include, for example, chemotherapeutic drugs such asvinca alkaloids, anthracyclines, epidophyllotoxins, taxanes,antimetabolites, alkylating agents, antibiotics, Cox-2 inhibitors,antimitotics, antiangiogenic and proapoptotic agents, particularlydoxorubicin, methotrexate, taxol, CPT-11, camptothecans, and others fromthese and other classes of anticancer agents, and the like. Other cancerchemotherapeutic drugs include nitrogen mustards, alkyl sulfonates,nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purineanalogs, platinum coordination complexes, hormones, and the like.Suitable chemotherapeutic agents are described in REMINGTON'SPHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and inGOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed.(MacMillan Publishing Co. 1985), as well as revised editions of thesepublications. Other suitable chemotherapeutic agents, such asexperimental drugs, are known to those of skill in the art, and may beconjugated to the stably tethered structures described herein usingmethods that are known in the art.

Another class of therapeutic agents consists of radionuclides that emitα-particles (such as ²¹²Pb, ²¹²Bi, ²¹³Bi, ²¹¹At, ²²³Ra, ²²⁵Ac), βarticles (such as ³²P, ³³P, ⁴⁷Sc, ⁶⁷Cu, ⁶⁷Ga, ⁸⁹Sr, ⁹⁰Y, ¹¹¹Ag, ¹²⁵I,¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁶⁶Dy, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re), orAuger electrons (such as ¹¹¹In, ¹²⁵I, ⁶⁷Ga, ¹⁹¹Os, ^(193m)Pt, ^(195m)Pt,^(195m)Hg). The stably tethered structures may be labeled with one ormore of the above radionuclides using methods as described for thediagnostic agents.

A suitable peptide containing a detectable label (e.g., a fluorescentmolecule), or a cytotoxic agent, (e.g., a radioiodine), can becovalently, non-covalently, or otherwise associated with the stablytethered structures. For example, a therapeutically useful conjugate canbe obtained by incorporating a photoactive agent or dye onto the stablytethered structures. Fluorescent compositions, such as fluorochrome, andother chromogens, or dyes, such as porphyrins sensitive to visiblelight, have been used to detect and to treat lesions by directing thesuitable light to the lesion. In therapy, this has been termedphotoradiation, phototherapy, or photodynamic therapy. See Jori et al.(eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES (LibreriaProgetto 1985); van den Bergh, Chem. Britain (1986), 22:430. Moreover,monoclonal antibodies have been coupled with photoactivated dyes forachieving phototherapy. See Mew et al., J. Immunol. (1983), 130:1473;idem., Cancer Res. (1985), 45:4380; Oseroff et al., Proc. Natl. Acad.Sci. USA (1986), 83:8744; idem., Photochem. Photobiol. (1987), 46:83;Hasan et al., Prog. Clin. Biol. Res. (1989), 288:471; Tatsuta et al.,Lasers Surg. Med. (1989), 9:422; Pelegrin et al., Cancer (1991),67:2529. Endoscopic or laparoscopic applications are also contemplated.Endoscopic methods of detection and therapy are described in U.S. Pat.No. 4,932,412; U.S. Pat. No. 5,525,338; U.S. Pat. No. 5,716,595; U.S.Pat. No. 5,736,119; U.S. Pat. No. 5,922,302; U.S. Pat. No. 6,096,289;and U.S. Pat. No. 6,387,350, which are incorporated herein by referencein their entirety.

In certain embodiments, the novel constructs and methods disclosedherein are useful for targeted delivery of RNAi for therapeuticintervention. The delivery vehicle can be a stably tethered structurewith an internalizing antibody binding domain fused to human protamine(peptide of ˜50 amino acid residues) as its precursor. An example of ana₂ construct of use would be VH-CH1-hP1-DDD1//VL-CL orVH-CH1-hP2-DDD1//VL-CL, where hP1 and hP2 are human protamine 1 andhuman protamine 2, respectively; both capable of forming stable DNAcomplexes for in vivo applications (Nat Biotechnol. 23: 709-717, 2005;Gene Therapy. 13: 194-195, 2006). An example of an a₄ construct of usewould be VH-CH1-hP1-DDD2//VL-CL or VH-CH1-hP2-DDD2//VL-CL, which wouldprovide four active Fab fragments, each carrying a human protamine forbinding to RNAi. The multivalent complex will facilitate the binding toand receptor-mediated internalization into target cells, where thenoncovalently bound RNAi is dissociated in the endosomes and releasedinto cytoplasm. As no redox chemistry is involved, the existence of 3intramolecular disulfide bonds in hP1 or hP2 does not present a problem.In addition to delivery of RNAi, these constructs may also be of use fortargeted delivery of therapeutic genes or DNA vaccines. Another area ofuse is to apply the technology for producing intrabodies, which is theprotein analog of RNAi in terms of function.

Therapeutic Agents

Pharmaceutical Compositions

In some embodiments, a stably tethered structure and/or one or moreother therapeutic agents may be administered to a subject, such as asubject with cancer. Such agents may be administered in the form ofpharmaceutical compositions. Generally, this will entail preparingcompositions that are essentially free of impurities that could beharmful to humans or animals. One skilled in the art would know that apharmaceutical composition can be administered to a subject by variousroutes including, for example, orally or parenterally, such asintravenously.

In certain embodiments, an effective amount of a therapeutic agent mustbe administered to the subject. An “effective amount” is the amount ofthe agent that produces a desired effect. An effective amount willdepend, for example, on the efficacy of the agent and on the intendedeffect. For example, a lesser amount of an antiangiogenic agent may berequired for treatment of a hyperplastic condition, such as maculardegeneration or endometriosis, compared to the amount required forcancer therapy in order to reduce or eliminate a solid tumor, or toprevent or reduce its metastasizing. An effective amount of a particularagent for a specific purpose can be determined using methods well knownto those in the art.

Chemotherapeutic Agents

In certain embodiments, chemotherapeutic agents may be administered.Anti-cancer chemotherapeutic agents of use include, but are not limitedto, 5-fluorouracil, bleomycin, busulfan, camptothecins, carboplatin,chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin,daunorubicin, doxorubicin, estrogen receptor binding agents, etoposide(VP16), farnesyl-protein transferase inhibitors, gemcitabine,ifosfamide, mechlorethamine, melphalan, methotrexate, mitomycin,navelbine, nitrosurea, plicomycin, procarbazine, raloxifene, tamoxifen,taxol, temazolomide (an aqueous form of DTIC), transplatinum,vinblastine and methotrexate, vincristine, or any analog or derivativevariant of the foregoing. Chemotherapeutic agents of use againstinfectious organisms include, but are not limited to, acyclovir,albendazole, amantadine, amikacin, amoxicillin, amphotericin B,ampicillin, aztreonam, azithromycin, bacitracin, bactrim, Batrafen®,bifonazole, carbenicillin, caspofungin, cefaclor, cefazolin,cephalosporins, cefepime, ceftriaxone, cefotaxime, chloramphenicol,cidofovir, Cipro®, clarithromycin, clavulanic acid, clotrimazole,cloxacillin, doxycycline, econazole, erythrocycline, erythromycin,flagyl, fluconazole, flucytosine, foscarnet, furazolidone, ganciclovir,gentamycin, imipenem, isoniazid, itraconazole, kanamycin, ketoconazole,lincomycin, linezolid, meropenem, miconazole, minocycline, naftifine,nalidixic acid, neomycin, netilmicin, nitrofurantoin, nystatin,oseltamivir, oxacillin, paromomycin, penicillin, pentamidine,piperacillin-tazobactam, rifabutin, rifampin, rimantadine, streptomycin,sulfamethoxazole, sulfasalazine, tetracycline, tioconazole, tobramycin,tolciclate, tolnaftate, trimethoprim sulfamethoxazole, valacyclovir,vancomycin, zanamir, and zithromycin.

Chemotherapeutic agents and methods of administration, dosages, etc.,are well known to those of skill in the art (see for example, the“Physicians Desk Reference”, Goodman & Gilman's “The PharmacologicalBasis of Therapeutics” and in “Remington's Pharmaceutical Sciences”,incorporated herein by reference in relevant parts). Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.

Hormones

Corticosteroid hormones can increase the effectiveness of otherchemotherapy agents, and consequently, they are frequently used incombination treatments. Prednisone and dexamethasone are examples ofcorticosteroid hormones. Progestins, such as hydroxyprogesteronecaproate, medroxyprogesterone acetate, and megestrol acetate, have beenused in cancers of the endometrium and breast. Estrogens such asdiethylstilbestrol and ethinyl estradiol have been used in cancers suchas prostate cancer. Antiestrogens such as tamoxifen have been used incancers such as breast cancer. Androgens such as testosterone propionateand fluoxymesterone have also been used in treating breast cancer.

Angiogenesis Inhibitors

In certain embodiments, anti-angiogenic agents, such as angiostatin,baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-P1GFpeptides and antibodies, anti-vascular growth factor antibodies,anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, lamininpeptides, fibronectin peptides, plasminogen activator inhibitors, tissuemetalloproteinase inhibitors, interferons, interleukin-12, IP-10, Gro-β,thrombospondin, 2-methoxyoestradiol, proliferin-related protein,carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate,angiopoietin-2, interferon-alpha, herbimycin A, PNU145156E, 16Kprolactin fragment, Linomide, thalidomide, pentoxifylline, genistein,TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir,vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline maybe of use.

Immunomodulators

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins and hematopoietic factors, such asinterleukins, colony-stimulating factors, interferons (e.g.,interferons-α, -β and -γ) and the stem cell growth factor designated “S1factor.” Examples of suitable immunomodulator moieties include IL-2,IL-6, IL-10, IL-12, IL-18, IL-21, interferon-gamma, TNF-alpha, and thelike.

The term “cytokine” is a generic term for proteins or peptides releasedby one cell population which act on another cell as intercellularmediators. As used broadly herein, examples of cytokines includelymphokines, monokines, growth factors and traditional polypeptidehormones. Included among the cytokines are growth hormones such as humangrowth hormone, N-methionyl human growth hormone, and bovine growthhormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; glycoprotein hormones such as follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);hepatic growth factor; prostaglandin, fibroblast growth factor;prolactin; placental lactogen, OB protein; tumor necrosis factor-α and-β; mullerian-inhibiting substance; mouse gonadotropin-associatedpeptide; inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-21, LIF, G-CSF, GM-CSF, M-CSF, EPO, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand LT. As used herein, the term cytokine includes proteins from naturalsources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

Chemokines generally act as chemoattractants to recruit immune effectorcells to the site of chemokine expression. Chemokines include, but arenot limited to, RANTES, MCAF, MIP1-alpha, MIP1-Beta, and IP-10. Theskilled artisan will recognize that certain cytokines are also known tohave chemoattractant effects and could also be classified under the termchemokines. Similarly, the terms immunomodulator and cytokine overlap intheir respective members.

Radioisotope Therapy and Radioimmunotherapy

In some embodiments, the peptides and/or proteins may be of use inradionuclide therapy or radioimmunotherapy methods (see, e.g., Govindanet al., 2005, Technology in Cancer Research & Treatment, 4:375-91;Sharkey and Goldenberg, 2005, J. Nucl. Med. 46:115S-1275; Goldenberg etal. (J Clin Oncol 2006; 24:823-834), “Antibody Pre-targeting AdvancesCancer Radioimmunodetection and Radioimmunotherapy,” each incorporatedherein by reference.) In specific embodiments, stably tetheredstructures may be directly tagged with a radioisotope of use andadministered to a subject. In alternative embodiments, radioisotope(s)may be administered in a pre-targeting method as discussed above, usinga haptenic peptide or ligand that is radiolabeled and injected afteradministration of a bispecific stably tethered structure that localizesat the site of elevated expression in the diseased tissue.

Radioactive isotopes useful for treating diseased tissue include, butare not limited to-¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁷Cu, ⁹⁰Y,¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy,¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr,⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, and ²¹¹Pb.The therapeutic radionuclide preferably has a decay energy in the rangeof 20 to 6,000 keV, preferably in the ranges 60 to 200 keV for an Augeremitter, 100-2,500 keV for a beta emitter, and 4,000-6,000 keV for analpha emitter. Maximum decay energies of useful beta-particle-emittingnuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV, andmost preferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255. Decay energies ofuseful alpha-particle-emitting radionuclides are preferably 2,000-10,000keV, more preferably 3,000-8,000 keV, and most preferably 4,000-7,000keV.

For example, ⁶⁷Cu, considered one of the more promising radioisotopesfor radioimmunotherapy due to its 61.5 hour half-life and abundantsupply of beta particles and gamma rays, can be conjugated to a proteinor peptide using the chelating agent,p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid (TETA).Alternatively, ⁹⁰Y, which emits an energetic beta particle, can becoupled to a peptide, antibody, fusion protein, or fragment thereof,using diethylenetriaminepentaacetic acid (DTPA).

Additional potential radioisotopes include ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br, ¹⁹⁸Au,²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg,²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm, ¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁹⁷Pt,¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ⁵⁹Fe,⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.

In another embodiment, a radiosensitizer can be used. The addition ofthe radiosensitizer can result in enhanced efficacy. Radiosensitizersare described in D. M. Goldenberg (ed.), CANCER THERAPY WITHRADIOLABELED ANTIBODIES, CRC Press (1995), which is incorporated hereinby reference in its entirety.

The stably tethered structure that has a boron addend-loaded carrier forthermal neutron activation therapy will normally be effective in someways. However, it will be advantageous to wait until non-targetedimmunoconjugate clears before neutron irradiation is performed.Clearance can be accelerated using an antibody that binds to the ligand.See U.S. Pat. No. 4,624,846 for a description of this general principle.For example, boron addends such as carboranes, can be attached toantibodies. Carboranes can be prepared with carboxyl functions onpendant side chains, as is well-known in the art. Attachment ofcarboranes to a carrier, such as aminodextran, can be achieved byactivation of the carboxyl groups of the carboranes and condensationwith amines on the carrier. The intermediate conjugate is thenconjugated to the antibody. After administration of the conjugate, aboron addend is activated by thermal neutron irradiation and convertedto radioactive atoms which decay by alpha-emission to produce highlytoxic, short-range effects.

Kits

Various embodiments may concern kits containing components suitable fortreating or diagnosing diseased tissue in a patient. Exemplary kits maycontain at least one stably tethered structure. If the compositioncontaining components for administration is not formulated for deliveryvia the alimentary canal, such as by oral delivery, a device capable ofdelivering the kit components through some other route may be included.One type of device, for applications such as parenteral delivery, is asyringe that is used to inject the composition into the body of asubject. Inhalation devices may also be used.

The kit components may be packaged together or separated into two ormore separate containers. In some embodiments, the containers may bevials that contain sterile, lyophilized formulations of a compositionthat are suitable for reconstitution. A kit may also contain one or morebuffers suitable for reconstitution and/or dilution of other reagents.Other containers that may be used include, but are not limited to, apouch, tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

Formulation and Administration

The stably tethered structures, including their conjugates, may befurther formulated to obtain compositions that include one or morepharmaceutically suitable excipients, one or more additionalingredients, or some combination of these. These can be accomplished byknown methods to prepare pharmaceutically useful dosages, whereby theactive ingredients (i.e., the stably tethered structures or conjugates),are combined in a mixture with one or more pharmaceutically suitableexcipients. Sterile phosphate-buffered saline is one example of apharmaceutically suitable excipient. Other suitable excipients are wellknown to those in the art. See, e.g., Ansel et al., PHARMACEUTICALDOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18thEdition (Mack Publishing Company 1990), and revised editions thereof.

The preferred route for administration of the compositions describedherein is parental injection. In parenteral administration, thecompositions will be formulated in a unit dosage injectable form such asa solution, suspension or emulsion, in association with apharmaceutically acceptable excipient. Such excipients are inherentlynontoxic and nontherapeutic. Examples of such excipients are saline,Ringer's solution, dextrose solution and Hank's solution. Nonaqueousexcipients such as fixed oils and ethyl oleate may also be used. Apreferred excipient is 5% dextrose in saline. The excipient may containminor amounts of additives such as substances that enhance isotonicityand chemical stability, including buffers and preservatives. Othermethods of administration, including oral administration, are alsocontemplated.

Formulated compositions comprising stably tethered structures can beused for intravenous administration via, for example, bolus injection orcontinuous infusion. Compositions for injection can be presented in unitdosage form, e.g., in ampules or in multi-dose containers, with an addedpreservative. Compositions can also take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the compositions can be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compositions may be administered in solution. The pH of the solutionshould be in the range of pH 5 to 9.5, preferably pH 6.5 to 7.5. Theformulation thereof should be in a solution having a suitablepharmaceutically acceptable buffer such as phosphate, tris(hydroxymethyl)aminomethane-HCl or citrate and the like. Bufferconcentrations should be in the range of 1 to 100 mM. The formulatedsolution may also contain a salt, such as sodium chloride or potassiumchloride in a concentration of 50 to 150 mM. An effective amount of astabilizing agent such as glycerol, albumin, a globulin, a detergent, agelatin, a protamine or a salt of protamine may also be included.Systemic administration of the formulated composition is typically madeevery two to three days or once a week if a humanized form of theantibody is used as a template for the stably tethered structures.Alternatively, daily administration is useful. Usually administration isby either intramuscular injection or intravascular infusion.

The compositions may be administered to a mammal subcutaneously or byother parenteral routes. Moreover, the administration may be bycontinuous infusion or by single or multiple boluses. Methods useful forthe antibodies or immunoconjugates can be applied to the compositionsdescribed herein. In general, the dosage of an administeredimmunoconjugate, fusion protein or naked antibody for humans will varydepending upon such factors as the patient's age, weight, height, sex,general medical condition and previous medical history. Typically, it isdesirable to provide the recipient with a dosage of the activeingredient that is in the range of from about 1 mg/kg to 20 mg/kg as asingle intravenous infusion, although a lower or higher dosage also maybe administered as circumstances dictate. This dosage may be repeated asneeded, for example, once per week for 4-10 weeks, preferably once perweek for 8 weeks, and more preferably, once per week for 4 weeks. It mayalso be given less frequently, such as every other week for severalmonths. The dosage may be given through various parenteral routes, withappropriate adjustment of the dose and schedule. In various exemplaryembodiments, dosages may range from 100 to 500 mg, from 200 to 1000 mg,from 500 to 2000 mg, from 100 to 250 mg, from 250 to 500 mg, from 500 to1000 mg, or other ranges known for antibody, antibody fragment or fusionprotein administration.

Pharmaceutical methods employed to control the duration of action ofimmunoconjugates or antibodies may be applied to the formulatedcompositions described herein. Control release preparations can beachieved through the use of biocompatible polymers to complex or adsorbthe immunoconjugate or naked antibody, for example, matrices ofpoly(ethylene-co-vinyl acetate) and matrices of a polyanhydridecopolymer of a stearic acid dimer and sebacic acid. See Sherwood et al.,Bio/Technology (1992), 10: 1446. The rate of release of animmunoconjugate or antibody from such a matrix depends upon themolecular weight of the immunoconjugate or antibody, the amount ofimmunoconjugate, antibody within the matrix, and the size of dispersedparticles. See Saltzman et al., Biophys. J (1989), 55: 163; Sherwood etal., supra. Other solid dosage forms are described in Ansel et al.,PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea& Febiger 1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES,18th Edition (Mack Publishing Company 1990), and revised editionsthereof.

For purposes of therapy, the composition is administered to a mammal ina therapeutically effective amount. A suitable subject for thetherapeutic and diagnostic methods disclosed herein is usually a human,although a non-human animal subject, such as mammals, cats, dogs,horses, pigs, goats, cows, alpacas, llamas or sheep is alsocontemplated.

The stably tethered structures disclosed herein are particularly usefulin the method of treating autoimmune disorders, disclosed in pendingU.S. Ser. No. 09/590,284 filed on Jun. 9, 2000 entitled “Immunotherapyof Autoimmune Disorders using Antibodies that Target B-Cells,” which isincorporated in its entirety by reference. Compositions containing suchbinding structures are preferably administered intravenously orintramuscularly at a dose of 20-5000 mg. Administration may also beintranasal or by other nonparenteral routes. The compositions may alsobe administered via microspheres, liposomes or other microparticulatedelivery systems placed in certain tissues including blood.

The compositions may be administered by aerosol to achieve localizeddelivery to the lungs. Either an aqueous aerosol or a nonaqueous (e.g.,fluorocarbon propellent) suspension could be used. Sonic nebulizerspreferably are used in preparing aerosols to minimize exposing thestably tethered structure in the compositions to shear, which can resultin its degradation and loss of activity.

In general, the dosage of administration will vary depending upon suchfactors as the patient's age, weight, height, sex, general medicalcondition and previous medical history. Preferably, a saturating dose ofthe stably tethered structure is administered to a patient.

Typically, it is desirable to provide the recipient with a dosage thatis in the range of from about 50 to 500 milligrams of the stablytethered structure, although a lower or higher dosage also may beadministered as circumstances dictate. Examples of dosages include 20 to1500 milligrams protein per dose, 20 to 500 milligrams protein per dose,20 to 100 milligrams protein per dose, 20 to 1000 milligrams protein perdose, 100 to 1500 milligrams protein per dose. In the embodiments wherethe composition comprises a radionuclide, the dosage may be measured bymillicurries. In the case of ⁹⁰Y, the dosage may be between 15 and 40mCi, 10 and 30 mCi, 20 and 30 mCi, or 10 and 20 mCi.

A stably tethered structure linked to a radionuclide is particularlyeffective for microbial therapy. After it has been determined that thestably tethered structure is localized at one or more infectious sitesin a subject, higher doses of the labeled composition, generally from 20mCi to 150 mCi per dose for ¹³¹I, 5 mCi to 30 mCi per dose for ⁹⁰Y, or 5mCi to 20 mCi per dose of ¹⁸⁶Re, each based on a 70 kg patient weight,are injected. Injection may be intravenous, intraarterial,intralymphatic, intrathecal, or intracavitary (i.e., parenterally), andmay be repeated. It may be advantageous for some therapies to administermultiple, divided doses, thus providing higher microbial toxic doseswithout usually effecting a proportional increase in radiation of normaltissues.

Chemotherapeutic agents, antimicrobial agents, cytokines,granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage-colony stimulating factor (GM-CSF), erythropoietin,thrombopoietin, and the like, which are not chemically linked to thestably tethered structures, may be administered before, during, or afterthe administration of the composition. Alternatively, such agents may beattached to the stably tethered structures.

The stably tethered structures in the a₂b format are particularlysuitable as pretargeting agents. A exemplary structure will consist oftwo scFv or Fab subunits as a₂ that bind bivalently to a target tissueor cell, and one scFv or Fab subunit as b that binds to a hapten. Such abispecific trivalent structure is first administered to a subject,optionally followed by a clearing agent, followed by administration ofan agent in which the hapten is bound to a functional agent, such as adetectable label for diagnosis, or a therapeutic agent for methods oftreatment. The skilled artisan will be aware that other known methods ofusing bispecific antibodies may also be practiced using the stablytethered structures. These methods of diagnosis and therapy may beapplied in essentially any circumstance in which antibody-based agentshave been used for diagnosis or therapy. As discussed below, bispecifichexavalent stably tethered structures may also be utilized for the samepurposes as bispecific trivalent structures.

Uses for Treatment and Diagnosis: Applications not InvolvingPretargeting

The stably tethered structures, including their conjugates, are suitablefor use in a wide variety of therapeutic and diagnostic applicationsthat utilize antibodies or immunoconjugates and do not requirepretargeting. For example, the trivalent structures can be used fortherapy as a “naked” construct, i.e. in an embodiment where such astructure is not conjugated to an additional functional agent, in thesame manner as therapy using a naked antibody. Alternatively, the stablytethered structures can be derivatized with one or more functionalagents to enable diagnostic or therapeutic applications. The additionalagent may be covalently linked to the stably tethered structures asdescribed above.

Also contemplated is the use of radioactive and non-radioactivediagnostic agents, which are linked to the stably tethered structures.Suitable non-radioactive diagnostic agents are those used for magneticresonance imaging (MRI), computed tomography (CT) or ultrasound. MRIagents include, for example, non-radioactive metals, such as manganese,iron and gadolinium, which are complexed with suitable chelates such as2-benzyl-DTPA and its monomethyl and cyclohexyl analogs. See U.S. Ser.No. 09/921,290 filed on Oct. 10, 2001, which is incorporated in itsentirety by reference.

The stably tethered structures may be labeled with a radioisotope usefulfor diagnostic imaging. Suitable radioisotopes may include those in theenergy range of 60 to 4,000 KeV, or more specifically, ¹⁸F, ⁵²Fe, ⁶²Cu,⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ⁴⁵Ti,¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ¹⁷⁷Lu, ³²P, ¹⁸⁸Re, and thelike, or a combination thereof. See, e.g., U.S. patent applicationentitled “Labeling Targeting Agents with Gallium-68”-Inventors G. L.Griffiths and W. J. McBride, and U.S. Provisional Application No.60/342,104, which discloses positron emitters, such as ¹⁸F, ⁶⁸Ga,^(94m)Tc, and the like, for imaging purposes; incorporated entirely byreference). Detection can be achieved, for example, by single photonemission computed tomography (SPECT), or positron emission tomography(PET). The application also may be for intraoperative diagnosis toidentify occult neoplastic tumors.

In another embodiment the stably tethered structures may be labeled withone or more radioactive isotopes useful for killing neoplastic or otherrapidly dividing cells, which include β-emitters (such as ³²P, ³³P,⁴⁷Sc, ⁶⁷Cu, ⁶⁷Ga, ⁸⁹Sr, ⁹⁰Y, ¹¹¹Ag, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb,¹⁶⁶Ho, ¹⁶⁶Dy, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re), Auger electron emitters suchas ¹¹¹In, ¹²⁵I, ⁶⁷Ga, ¹⁹¹Os, ^(193m)Pt, ^(195m)Pt, ^(195m)Hg),α-emitters (such as ²¹²Pb, ²¹²Bi, ²¹³Bi, ²¹¹At, ²²³Ra, ²²⁵Ac), or acombination thereof.

The stably tethered structures may be used for MRI by linking to one ormore image enhancing agents, which may include complexes of metalsselected from the group consisting of chromium (III), manganese (II),iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) and erbium (III).Similarly, the stably tethered structures may be used for ultrasoundimaging by linking to one or more image enhancing agents currently onthe market. U.S. Pat. No. 6,331,175 describes MRI technique and thepreparation of antibodies conjugated to an MRI enhancing agent and isincorporated in its entirety by reference.

A functional protein, such as a toxin, may be present in the stablytethered structures in several ways. For example, a functional proteinmay serve as the precursor for either component of the binary complex byfusing to either DDD2 or AD2, which is then combined with a targetingentity, composed of, for example, Fab/AD2 or Fab/DDD2, respectively.Alternatively, a functional protein can be fused to a targetingstructure to serve as a precursor for A, and the resulting A isoptionally paired with a suitable B. Toxins that may be used in thisregard include ricin, abrin, ribonuclease (RNase), DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. (See,e.g., Pastan. et al., Cell (1986), 47:641, and Goldenberg, C A—A CancerJournal for Clinicians (1994), 44:43. Additional toxins suitable for useherein are known to those of skill in the art and are disclosed in U.S.Pat. No. 6,077,499, which is incorporated in its entirety by reference.Other functional proteins of interest include various cytokines,clot-dissolving agents, enzymes, and fluorescent proteins.

Also provided is a method of treating a neoplastic disorder in asubject, by administering to the subject a “naked” stably tetheredbinding structure as described above, where at least one of the antigenbinding sites binds to an antigen selected from the group consisting ofcarbonic anydrase IX, alpha-fetoprotein, A3, antigen specific for A33antibody, Ba 733, BrE3-antigen, CA125, carcinoembryonic antigen(CEACAM5), CEACAM6, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21,CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD138,colon-specific antigen-p (CSAp), EGFR, EGP-1, EGP-2, Flt-1, Flt-3,folate receptor, HER2/neu, HLA-DR, human chorionic gonadrotropin, Ia,IL-2, IL-6, IL-8, insulin-like growth factor, KC4-antigen, KS-1, KS1-4,Le(y), macrophage-inhibition factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4,NCA66, NCA95, NCA90, necrosis antigens, antigen bound by p53, PAM-4antibody, placental growth factor, prostatic acid phosphatase, PSA,PSMA, RS5, S100, T101, TAC, TAG-72, Tn antigen, Thomson-Friedenreichantigens, tumor necrosis antigens, tenascin, TRAIL receptors, ED-Bfibronectin, VEGF, 17-1A-antigen, an angiogenesis marker, an oncogenemarker or an oncogene product. Antibodies against TRAIL receptors, suchas TRAIL-R1 and TRAIL-R2, are well known in the art. (See, e.g.,Georgakis et al., Br. J. Haematol. 2005, 130:501-510; Mori et al., FEBSLett. 2005, 579:5379-84.) Such antibodies or fragments may be used aloneor in combination with anti-TAA antibodies for cancer therapy.

The neoplastic disorder may be selected from the group consisting ofcarcinomas, sarcomas, gliomas, lymphomas, leukemias, and melanomas.Exemplary types of tumors that may be targeted include acutelymphoblastic leukemia, acute myelogenous leukemia, biliary cancer,breast cancer, cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, colorectal cancer, endometrial cancer, esophageal,gastric, head and neck cancer, Hodgkin's lymphoma, lung cancer,medullary thyroid, non-Hodgkin's lymphoma, ovarian cancer, pancreaticcancer, glioma, melanoma, liver cancer, prostate cancer, and urinarybladder cancer.

Also provided is a method for treating a B-cell malignancy, or B-cellimmune or autoimmune disorder in a subject, by administering to thesubject one or more dosages of a therapeutic composition containing astably tethered binding structure as described above and apharmaceutically acceptable carrier, where each antigen binding sitebinds a distinct epitope of CD19, CD20, CD22 or IL-17. The therapeuticcomposition may be parenterally administered in a dosage of 20 to 1500milligrams protein per dose, or 20 to 500 milligrams protein per dose,or to 100 milligrams protein per dose. The subject may receive repeatedparenteral dosages of 20 to 100 milligrams protein per dose, or repeatedparenteral dosages of 20 to 1500 milligrams protein per dose. In thesemethods, a sub-fraction of the binding structure may be labeled with aradioactive isotope, such as ³²P, ³³P, ⁴⁷Sc, ⁶⁷Cu, ⁶⁷Ga, ⁹⁰Y, ¹¹¹Ag,¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²¹¹At, ²²³Ra, and ²²⁵Ac, or acombination thereof.

Also provided is a method for detecting or diagnosing a B-cellmalignancy, or B-cell immune or autoimmune disorder in a subject, byadministering to the subject a diagnostic composition containing astably tethered binding structure, where each antigen binding site bindsa distinct epitope of CD19, CD20, CD22 or IL-17, a pharmaceuticallyacceptable carrier, and a radionuclide selected from the groupconsisting of ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr,^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹²⁵⁴⁻¹⁵⁸Gd,¹⁷⁷Lu, ³²P, ⁴⁵Ti and ¹⁸⁸Re, or a combination thereof. Detection may beby SPECT or PET as described above. The application also may be forintraoperative diagnosis to identify occult neoplastic tumors.

Also provided is a method for detecting or diagnosing a B-cellmalignancy, or B-cell immune or autoimmune disorder in a subject, byadministering to the subject a diagnostic composition containing astably tethered binding structure, where each antigen binding site bindsa distinct epitope of CD19, CD20, CD22 or IL-17, a pharmaceuticallyacceptable carrier, and one or more image enhancing agents for use inmagnetic resonance imaging (MRI). The image enhancing agent may beselected from those described above.

Also provided is a method of diagnosing and/or treating a non-neoplasticdisease or disorder, by administering to a subject suffering from thedisease or disorder a stably tethered binding structure, where adetectable label or therapeutic agent is attached, and where one or moreof the antigen binding sites is specific for a marker substance of thedisease or disorder. The disease or disorder may be caused by a fungus,such as Microsporum, Trichophyton, Epidermophyton, Sporothrix schenckii,Cryptococcus neoformans, Coccidioides immitis, Histoplasma capsulatum,Blastomyces dermatitidis, and Candida albicans, or a virus, such ashuman immunodeficiency virus (HIV), herpes virus, cytomegalovirus,rabies virus, influenza virus, human papilloma virus, hepatitis B virus,Sendai virus, feline leukemia virus, Reo virus, polio virus, human serumparvo-like virus, simian virus 40, respiratory syncytial virus, mousemammary tumor virus, Varicella-Zoster virus, Dengue virus, rubellavirus, measles virus, adenovirus, human T-cell leukemia viruses,Epstein-Barr virus, murine leukemia virus, mumps virus, vesicularstomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, andblue tongue virus.

The disease or disorder may be caused by a bacterium, such as Anthraxbacillus, Streptococcus agalactiae, Legionella pneumophilia,Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae,Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponemapallidum, Lyme disease spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, and Mycobacterium tuberculosis,or a Mycoplasma. The disease or disorder may be caused by a parasite,such as malaria.

The disease or disorder may be an autoimmune disease, such as acuteidiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenicpurpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemiclupus erythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcalnephritis, erythema nodosurn, Takayasu'sarteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitisubiterans, Sjogren's syndrome, primary biliary cirrhosis,Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic activehepatitis, polymyositis/dermatomyositis, polychondritis, parnphigusvulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophiclateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia,pernicious anemia, rapidly progressive glomerulonephritis, psoriasis,and fibrosing alveolitis.

The disease or disorder may be myocardial infarction, ischemic heartdisease, or atherosclerotic plaques, or graft rejection, or Alzheimer'sdisease, or caused by atopic tissue. The disease or disorder may beinflammation caused by accretion of activated granulocytes, monocytes,lymphoid cells or macrophages at the site of inflammation, and where theinflammation is caused by an infectious agent.

In addition, cells expressing a particular receptor or overexpressing areceptor may be targeted using a stably tethered structure whereineither the A or B component contains a ligand for the receptor thatdirects binding of the structure to the cell(s) bearing the receptor.Therapeutic or diagnostic agents can be fused or conjugated to one ormore of the subunits of the structure to permit methods of diagnosis andtherapy.

Uses for Treatment and Diagnosis: Applications Involving Pretargeting

Pretargeting is a multistep process originally developed to resolve theslow blood clearance of directly targeting antibodies, which contributesto undesirable toxicity to normal tissues, in particular, bone marrow.With pretargeting, a radionuclide or other therapeutic agent is attachedto a small compound that is cleared within minutes from the blood. Thepretargeting agent, which is capable of recognizing the smallradiolabeled compound in addition to the target antigen, is administeredfirst, and the radiolabeled compound is administered at a later timewhen the pretargeting agent is sufficiently cleared from the blood.

Pretargeting methods have been developed to increase thetarget:background ratios of detection or therapeutic agents. Examples ofpre-targeting and biotin/avidin approaches are described, for example,in Goodwin et al., U.S. Pat. No. 4,863,713; Goodwin et al., J. Nucl.Med. 29:226, 1988; Hnatowich et al., J. Nucl. Med. 28:1294, 1987; Oehret al., J. Nucl. Med. 29:728, 1988; Klibanov et al., J. Nucl. Med.29:1951, 1988; Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos etal., J. Nucl. Med. 31:1791, 1990; Schechter et al., Int. J. Cancer48:167, 1991; Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli etal., Nucl. Med. Commun. 12:211, 1991; U.S. Pat. No. 5,256,395; Stickneyet al., Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119,1991; U.S. Pat. No. 6,077,499; U.S. Ser. No. 09/597,580; U.S. Ser. No.10/361,026; U.S. Ser. No. 09/337,756; U.S. Ser. No. 09/823,746; U.S.Ser. No. 10/116,116; U.S. Ser. No. 09/382,186; U.S. Ser. No. 10/150,654;U.S. Pat. No. 6,090,381; U.S. Pat. No. 6,472,511; U.S. Ser. No.10/114,315; U.S. Provisional Application No. 60/386,411; U.S.Provisional Application No. 60/345,641; U.S. Provisional Application No.60/3328,835; U.S. Provisional Application No. 60/426,379; U.S. Ser. No.09/823,746; U.S. Ser. No. 09/337,756; U.S. Provisional Application No.60/342,103; and U.S. Pat. No. 6,962,702, all of which are incorporatedherein by reference.

In a specific, non-limiting example, a pretargeting agent based on thestably tethered structure contains two identical tumor antigen bindingsites that are specific for CEA and the third binding site is specificfor the hapten, histamine-succinyl-glycine (HSG). In alternativeembodiments, a different tumor-associated antigen may be targeted, withthe same or a different hapten.

For pretargeting applications, the targetable agent may be a liposomewith a bivalent HSG-peptide covalently attached to the outside surfaceof the liposome lipid membrane. The liposome may be gas filled forcontrast or may be filled with a therapeutic or diagnostic agent.

A pretargeting method of treating or diagnosing a disease or disorder ina subject is provided by (1) administering to the subject a bispecifictrivalent or hexavalent binding structure described above, where thefirst antigen binding sites are directed to a marker substance, ormarker substances specific for the disorder, and the second antigenbinding sites are directed to a targetable construct containing abivalent hapten; (2) optionally administering to the subject a clearingcomposition, and allowing the composition to clear the binding structurefrom circulation; and (3) administering to the subject the targetableconstruct containing a bivalent hapten, where the targetable constructfurther contains one or more chelated or chemically bound therapeutic ordiagnostic agents. The disease or disorder may be as described above.

Also provided is a method of antibody dependent enzyme prodrug therapy(ADEPT) by (1) administering to a patient with a neoplastic disorder abinding structure as above, where the structure contains a covalentlyattached enzyme capable of activating a prodrug, (2) optionallyadministering to the subject a clearing composition, and allowing thecomposition to clear the binding structure from circulation, and (3)administering the prodrug to the patient.

Additional Uses

In general, the stably tethered structures may be substituted forantibody-based agents that have shown efficacy for treating cancers ornon-cancer diseases. It is well known that radioisotopes, drugs, andtoxins can be conjugated to antibodies or antibody fragments whichspecifically bind to markers produced by or associated with cancercells, and that such antibody conjugates can be used to target theradioisotopes, drugs or toxins to tumor sites to enhance theirtherapeutic efficacy and minimize side effects. Examples of these agentsand methods are reviewed in Wawrzynczak and Thorpe (in Introduction tothe Cellular and Molecular Biology of Cancer, L. M. Franks and N. M.Teich, eds, Chapter 18, pp. 378-410, Oxford University Press. Oxford,1986), in Immunoconjugates. Antibody Conjugates in Radioimaging andTherapy of Cancer (C. W. Vogel, ed., 3-300, Oxford University Press,N.Y., 1987), and in Dillman, R. O. (CRC Critical Reviews inOncology/Hematology 1:357, CRC Press, Inc., 1984). See also Pastan etal., Cell (1986), 47:641; Vitetta et al., Science (1987), 238:1098-1104;and Brady et al., Int. J. Rad. Oncol. Biol. Phys. (1987), 13:1535-1544.

In certain embodiments, multivalent stably tethered structures may be ofuse in treating and/or imaging normal or diseased tissue and organs, forexample using the methods described in U.S. Pat. Nos. 6,126,916;6,077,499; 6,010,680; 5,776,095; 5,776,094; 5,776,093; 5,772,981;5,753,206; 5,746,996; 5,697,902; 5,328,679; 5,128,119; 5,101,827; and4,735,210, each incorporated herein by reference. Additional methods aredescribed in U.S. application Ser. No. 09/337,756 filed Jun. 22, 1999and in U.S. application Ser. No. 09/823,746, filed Apr. 3, 2001. Suchimaging can be conducted by direct labeling of the stably tetheredstructure, or by a pretargeted imaging method, as described inGoldenberg et al, “Antibody Pretargeting Advances CancerRadioimmunodetection and Radiotherapy,” (in press, J. Clin. Oncol.), seealso U.S. Patent Publication Nos. 20050002945, 20040018557, 20030148409and 20050014207, each incorporated herein by reference.

Other examples of the use of immunoconjugates for cancer and other formsof therapy have been disclosed, inter alia, in the following U.S. Pat.Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457, 4,444,744, 4,460,459,4,460,561 4,624,846, 4,818,709, 4,046,722, 4,671,958, 4,046,784,5,332,567, 5,443,953, 5,541,297, 5,601,825, 5,635,603, 5,637,288,5,677,427, 5,686,578, 5,698,178, 5,789,554, 5,922,302, 6,187,287, and6,319,500. These methods are also applicable to the methods disclosedherein by the substitution of the engineered antibodies and antibodiesof the previous methods with the present stably tethered structures.

In some embodiments, the stably tethered structures disclosed andclaimed herein may be of use in radionuclide therapy orradioimmunotherapy methods (see, e.g., Govindan et al., 2005, Technologyin Cancer Research & Treatment, 4:375-91; Sharkey and Goldenberg, 2005,J. Nucl. Med. 46:115 S-127S; Goldenberg et al. (in press, J. Clin.Oncol.), “Antibody Pretargeting Advances Cancer Radioimmunodetection andRadioimmunotherapy,” each incorporated herein by reference.)

In another embodiment, a radiosensitizer can be used in combination witha naked or conjugated stably tethered structure, antibody or antibodyfragment. For example, the radiosensitizer can be used in combinationwith a radiolabeled stably tethered structure. The addition of theradiosensitizer can result in enhanced efficacy when compared totreatment with the radiolabeled stably tethered structure alone.Radiosensitizers are described in D. M. Goldenberg (ed.), CANCER THERAPYWITH RADIOLABELED ANTIBODIES, CRC Press (1995), which is incorporatedherein by reference in its entirety.

The stably tethered structures for use in any of the claimed methods,may be associated or administered with antimicrobial agents.

The stably tethered structure, for use in any of the claimed methods,may be associated or administered with cytokines and immune modulators.These cytokines and immune modulators, include, at least, interferons ofalpha, beta and gamma, and colony stimulating factors.

The disclosed methods may also be of use for stimulating the immuneresponse in a patient using the stably tethered structures. In oneembodiment, the stably tethered structure may comprise an antigenbinding site (ABS) of an anti-idiotype antibody. Such a stably tetheredstructure may mimic an epitope of a tumor-associated antigen to enhancethe body's immune response.

The stably tethered structure may be used for many immunologicalprocedures currently employing antibodies. These procedures include theuse of anti-idiotypic antibodies and epitope conjugated antibodies toboost the immune system. See U.S. Pat. Nos. 5,798,100; 6,090,381; and6,132,718. Anti-idiotypic antibodies are also employed as vaccinesagainst cancers and infectious diseases. See U.S. Pat. Nos. 6,440,416and 6,472,511. Further, a polyspecific trimeric or hexameric bindingstructure may bind multidrug transporter proteins and overcome multidrugresistant phenotype in cells and pathogens. The antibodies in thesemethods may be replaced by the stably tethered structure disclosedherein.

Various embodiments concern methods for treating a symptom of anautoimmune disorder. In the method, a stably tethered structure isadministered to a patient with an autoimmune disorder, which may beadmixed with a pharmaceutically acceptable carrier beforeadministration. The stably tethered structure of this method shouldcontain at least one ABS with binding specificity to a B-cell or T-cellantigen epitope. The B cell antigen may be CD22 and the epitope may beepitope A, epitope B, epitope C, epitope D and epitope E of CD22 andothers. The B cell-associated antigen may also be another cell antigensuch as CD 19, CD20, HLA-DR and CD74. The T-cell antigens may includeCD25. In certain embodiments, stably tethered structures of use to treatautoimmune disease may be selected to bind to IL-17.

The ABS may contain a sequence of subhuman primate, murine monoclonalantibody, chimeric antibody, humanized antibody, or human origin. Forexample, the ABS may be of humanized LL2 (anti-CD22), humanized LL1(anti-CD74) or humanized A20 (anti-CD20) monoclonal antibody origin.

The administration may be parenteral with dosages from 20 to 2000 mg perdose. Administration may be repeated until a degree of reduction insymptoms is achieved.

The patients who may be treated by the claimed methods include anyanimal including humans. Preferably, the animal is a mammal such ashumans, primates, equines, canines and felines.

The stably tethered structures may be used for the treatment of diseasesthat are resistant or refractory towards systemic chemotherapy. Theseinclude various viral, fungal, bacterial and protozoan infections, aswell as particular parasitic infections. Viral infections include thosecaused by influenza virus, herpes virus, Epstein-Barr virus andcytomegalovirus, rabies virus (Rhabdoviridae), papilloma virus, andpapovavirus, all of which are difficult to treat with systemicantibiotic/cytotoxic agents. Use of multivalent binding structures mayprovide a higher avidity for the target viruses, resulting insignificantly higher therapeutic index. Targeted radioimmunotherapyusing conjugates of the stably tethered structures that are labeled withradioisotopes (and including boron addends activatable with thermalneutron) offers a new approach to antiviral therapy.

Protozoans that may be treated by the methods described in the inventioninclude, e.g., Plasmodia (especially P. falciparum, the malariaparasite), Toxoplasma gondii (the toxoplasmosis infectious agent),Leishmaniae (infectious agent in leishmaniasis), and Escherichiahistolytica. Detection and treatment of malaria in its various stagesmay be significantly enhanced using the stably tethered structures.Monoclonal antibodies (mAbs) that bind to sporozoite antigens are known.However, since sporozoite antigens are not shared by blood stageparasites, the use of such mAbs against sporozoite antigens fortargeting is limited to a relatively short period of time in which thesporozoites are free in the circulation, just after injection and priorto development in the host's hepatocytes. Thus, it is preferable to usea mixture of mAbs that can target more than one parasite stage of aprotozoan (such as P. falciparum), which may be achieved with one ormore than one stably tethered structure having multiple specificity. Theuse of conjugates may offer further advantages for imaging, e.g. with^(99m)Tc, or for therapy, e.g., with ²¹¹At or an antimalarial drug,e.g., pyrimethamine.

Toxoplasmosis is also resistant to systemic chemotherapy. It is notclear whether mAbs that bind specifically to T. gondii, or natural, hostantibodies, can play a role in the immune response to toxoplasmosis but,as in the case of malarial parasites, appropriately targeted stablytethered structures may be effective vehicles for the delivery oftherapeutic agents.

Schistosomiasis, a widely prevalent helminth infection, is initiated byfree-swimming cercariae that are carried by some freshwater snails. Asin the case of malaria, there are different stages of cercariae involvedin the infectious process. Stably tethered structures that bind to aplurality of stages of cercariae, optionally to a plurality of epitopeson one or more thereof, and preferably in the form of a polyspecificcomposite, can be conjugated to an imaging or therapy agent foreffective targeting and enhanced therapeutic efficacy.

Stably tethered structures that bind to one or more forms of Trypanosomacruzi, the causative agent of Chagas' disease, can be made and used fordetection and treatment of this microbial infection. Stably tetheredstructures which react with a cell-surface glycoprotein or other surfaceantigens on differentiation stages of the trypanosome are suitable fordirecting imaging and therapeutic agents to sites of parasiticinfiltration in the body.

Another very difficult infectious organism to treat by available drugsis the leprosy bacillus (Mycobacterium leprae). Stably tetheredstructures that specifically bind to a plurality of epitopes on thesurface of M. leprae can be made and may be used, alone or incombination, to target imaging agents and/or antibiotic/cytotoxic agentsto the bacillus.

Helminthic parasitic infections, e.g., Strongyloidosis and Trichinosis,themselves relatively refractory towards chemotherapeutic agents, aresuitable targets for stably tethered structures. Their diagnosis andtherapy may be achieved by appropriate stably tethered structures orconjugates that bind specifically to one or, preferably, to a pluralityof epitopes on the parasites.

Antibodies are available or can easily be raised that specifically bindto most of the microbes and parasites responsible for the majority ofinfections in humans. Many of these have been used previously for invitro diagnostic purposes and may be incorporated into stably tetheredstructures as components of antibody conjugates to target diagnostic andtherapeutic agents to sites of infection. Microbial pathogens andinvertebrate parasites of humans and mammals are organisms with complexlife cycles having a diversity of antigens expressed at various stagesthereof. Therefore, targeted treatment can best be effected when stablytethered structures which recognize antigen determinants on thedifferent forms are made and used in combination, either as mixtures oras polyspecific conjugates, linked to the appropriate therapeuticmodality. The same principle applies to using the reagents comprisingstably tethered structures for detecting sites of infection byattachment of imaging agents, e.g., radionuclides and/or MRI enhancingagents.

Other embodiments concern methods of intraoperatively identifyingdiseased tissues by administering an effective amount of a stablytethered structure and a targetable construct where the stably tetheredstructure comprises at least one antigen binding site that specificallybinds a targeted tissue and at least one other antigen binding site thatspecifically binds the targetable construct; and wherein said at leastone antigen binding site is capable of binding to a complementarybinding moiety on the target cells, tissues or pathogen or on a moleculeproduced by or associated therewith.

Still other embodiments concern methods for the endoscopicidentification of diseased tissues, in a subject, by administering aneffective amount of a stably tethered structure and administering atargetable construct. The stably tethered structure comprises at leastone antigen binding site that specifically binds a targeted tissue andat least one antigen binding site that specifically binds the targetableconstruct; and wherein said at least one antigen binding site showsspecific binding to a complementary binding moiety on the target cells,tissues or pathogen or on a molecule produced by or associatedtherewith.

An alternative method of detection of use is wireless capsule endoscopy,using an ingested capsule camera/detector of the type that iscommercially available from, for example, Given Imaging (Norcross Ga.).Certain embodiments concern methods for the endoscopic identification ofdiseased tissues, in a subject, by administering an effective amount ofa stably tethered structure, and administering a targetable construct.In this embodiment, the stably tethered structure comprises at least oneantigen binding site that specifically binds a targeted tissue and atleast one antigen binding site that specifically binds the targetableconstruct; and wherein said at least one antigen binding site showsspecific binding to a complementary binding moiety on the target cells,tissues or pathogen or on a molecule produced by or associatedtherewith.

Alternative embodiments concern methods for the intravascularidentification of diseased tissues, in a subject by administering aneffective amount of a stably tethered structure and a targetableconstruct. The stably tethered structure comprises at least one antigenbinding site (ABS) that specifically binds a complementary bindingmoiety on the target cells, tissues or pathogen or on a moleculeproduced by or associated with the cell, tissues or pathogen, and atleast one ABS that specifically binds a targetable construct. The targettissue may be a normal tissue such as thyroid, liver, heart, ovary,thymus, parathyroid, endometrium, bone marrow, lymph nodes or spleen.

Some embodiments concern kits for practicing the claimed methods. Thekit may include a targetable construct. The targetable construct may belabeled by any of the agents described as suitable for targetableconstructs above. Further, the targetable construct may be unlabeled butthe kit may comprise labeling reagents to label the targetableconstruct. The labeling reagents, if included, may contain the label anda crosslinker. The kit may also contain a stably tethered structurecomprising at least one ABS specific for the targetable construct and atleast one ABS specific for a targetable tissue. The kit may optionallycontain a clearing composition to remove stably tethered structure fromcirculation.

Targets for Stably Tethered Structures

Additional disclosure concerning targets for stably tethered structuresare disclosed in provisional U.S. Patent Application Ser. No.60/634,076, “Methods and Compositions for Immunotherapy and Detection ofInflammatory and Immune-dysregulatory Disease, Infectious Disease,Pathologic Angiogenesis and Cancer,” by Goldenberg et al., filed Dec. 9,2004, the entire text of which is incorporated herein by reference.

In some embodiments, the stably tethered structures claimed herein reactspecifically with two different targets. The different targets mayinclude, but are not limited to, proinflammatory effectors of the innateimmune system, coagulation factors, complement factors and complementregulatory proteins, targets specifically associated with aninflammatory or immune-dysregulatory disorder, with an infectiouspathogen, or with a pathologic angiogenesis or cancer, wherein thislatter class of target is not a proinflammatory effector of the immunesystem or a coagulation factor. Thus, in certain embodiments the stablytethered structure contains at least one binding specificity related tothe diseased cell, pathologic angiogenesis or cancer, or infectiousdisease, and at least one specificity to a component of the immunesystem, such as a receptor or antigen of B cells, T cells, neutrophils,monocytes and macrophages, and dendritic cells, or modulators ofcoagulation, such as thrombin or tissue factor, or proinflammatorycytokines, such as IL-1, IL-6, IL-10, HMGB-1, and MIF.

The stably tethered structure can be naked, but can also be conjugatedto a diagnostic imaging agent (e.g., isotope, radiological contrastagent,) or to a therapeutic agent, including a radionuclide, a boroncompound, an immunomodulator, a peptide a hormone, a hormone antagonist,an enzyme, oligonucleotides, an enzyme inhibitor, a photoactivetherapeutic agent, a cytotoxic agent, an angiogenesis inhibitor, and acombination thereof. The binding of the stably tethered structure to atarget can down-regulate or otherwise affect an immune cell function,but the stably tethered structure also may bind to other targets that donot directly affect immune cell function. For example, ananti-granulocyte antibody, such as against CD66 or CEACAM6 (e.g., NCA90or NCA95), can be used to target granulocytes in infected tissues, andcan also be used to target cancers that express CEACAM6.

In one embodiment, the therapeutic agent is an oligonucleotide. Forexample, the oligonucleotide can be an antisense oligonucleotide, or adouble stranded interfering RNA (RNAi) molecule. The oligonucleotide canbe against an oncogene like bcl-2 or p53. An antisense moleculeinhibiting bcl-2 expression is described in U.S. Pat. No. 5,734,033. Itmay be conjugated to, or form the therapeutic agent portion of a stablytethered structure. Alternatively, the oligonucleotide may beadministered concurrently or sequentially with the stably tetheredstructure.

In another embodiment, the therapeutic agent is a boron addend, andtreatment entails irradiation with thermal or epithermal neutrons afterlocalization of the therapeutic agent. The therapeutic agent also may bea photoactive therapeutic agent, particularly one that is a chromogen ora dye.

In a preferred embodiment, the therapeutic agent is a cytotoxic agent,such as a drug or toxin. Also preferred, the drug is selected from thegroup consisting of nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas, gemcitabine, triazenes, folic acid analogs,anthracyclines, taxanes, COX-2 inhibitors, pyrimidine analogs, purineanalogs, antibiotics, enzymes, enzyme inhibitors, epipodophyllotoxins,platinum coordination complexes, vinca alkaloids, substituted ureas,methyl hydrazine derivatives, adrenocortical suppressants, hormoneantagonists, endostatin, taxols, SN38, camptothecins, doxorubicins andtheir analogs, antimetabolites, alkylating agents, antimitotics,antiangiogenic, apoptotoic agents, methotrexate, CPT-11, and acombination thereof.

In another preferred embodiment, the therapeutic agent is a toxinderived from a source selected from the group comprising an animal, aplant, and a microbial source. Preferred toxins include ricin, abrin,alpha toxin, saporin, ribonuclease (RNase), DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin,Pseudomonas exotoxin, and Pseudomonas endotoxins.

The therapeutic agent may be an immunomodulator, such as a cytokine, astem cell growth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), a stem cell growthfactor, erythropoietin, thrombopoietin and a combination thereof saidlymphotoxin is tumor necrosis factor (TNF). The hematopoietic factor maybe an interleukin (IL), the colony stimulating factor may be agranulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF)), the interferon may beinterferons-α, β or γ, and the stem cell growth factor may be S1 factor.Alternatively, the immunomodulator may comprise IL-1, IL-2, IL-3, IL-6,IL-10, IL-12, IL-17, IL-18, IL-21, interferon-γ, TNF-α, or a combinationthereof.

Preferred therapeutic radionuclides include beta, alpha, and Augeremitters, with a keV range of 80-500 keV. Exemplary therapeuticradioisotopes include ³²P, ³³P, ⁴⁷Sc, ¹²⁵I, ¹³¹I, ⁸⁶Y, ⁹⁰Y, ¹⁸⁶Re,¹⁸⁸Re, ¹⁸⁹Re, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ¹¹¹Ag, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy,¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁹⁸Au, ²¹¹At, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²³Ra and ²²⁵Ac, andcombinations thereof. Exemplary photoactive therapeutic agents areselected from the group comprising chromogens and dyes.

Still preferred, the therapeutic agent is an enzyme selected from thegroup comprising malate dehydrogenase, staphylococcal nuclease,delta-V-steroid isomerase, yeast alcohol dehydrogenase,α-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, β-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase.

Various examples of therapeutic agent peptides are known in the art andany such known agent may be used. Exemplary therapeutic peptidesinclude, but are not limited to, hormones, growth factors, cytokines,chemokines, binding peptides, blocking peptides, toxins, angiogenicfactors, anti-angiogenic factors, antibiotics, anti-cancer peptides,anti-viral peptides, pharmaceutical peptides, enzymes, agonists,antagonists, hematopoietic agents such as erythropoietin and many otherclinically useful compounds.

The stably tethered structure may bind specifically to at least oneproinflammatory effector cytokine, proinflammatory effector chemokine,or proinflammatory effector receptor. Proinflammatory effector cytokinesto which the stably tethered structure may bind include, but are notrestricted to, MIF, HMGB-1, TNF-α (tumor necrosis factor alpha), IL-1,IL-4, IL-5, IL-6, IL-8, IL-12, IL-15, IL-17 and IL-18. Proinflammatoryeffector chemokines include, but are not restricted to, CCL19, CCL21,IL-8, MCP-1 (monocyte chemotactic protein 1), RANTES, MIP-1A (macrophageinflammatory protein 1A), MIP-1B (macrophage inflammatory protein 1B),ENA-78 (epithelial neutrophil activating peptide 78), IP-10, GROB (GRObeta), and Eotaxin. Proinflammatory effector receptors include, but arenot restricted to, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R and IL-18R. Thestably tethered structure also may react specifically with at least onecoagulation factor, such as tissue factor or thrombin. Thelymphokines/cytokines react with their receptors on the immune cells toeffect activation, and antibodies can block activation by neutralizingthe lymphokine/cytokine. Alternatively, antibodies can react with thelymphokine/cytokine receptors to block activation.

The different targets to which the stably tethered structure bindsspecifically may be from the same or different classes of effectors andcoagulation factors. For example, the two or more different targets towhich the stably tethered structure binds specifically may be selectedfrom the same class of effectors or coagulation factors, such as two ormore different proinflammatory effector cytokines, two ore moredifferent proinflammatory effector chemokines, two or more differentproinflammatory effector receptors, or two or more coagulation factors.Alternatively, the two or more different targets may be selected fromdifferent classes of effectors and coagulation factors. For example, onetarget may be a proinflammatory effector of the innate immune system andone target may be a coagulation factor. Or the stably tethered structuremay react specifically with two different classes of proinflammatoryeffectors, such as at least one proinflammatory effector cytokine and atleast one proinflammatory effector chemokine, at least oneproinflammatory effector cytokine and at least one proinflammatoryeffector receptor, or at least one proinflammatory effector chemokineand at least one proinflammatory effector receptor. It may also be thecase that the two different targets with which the stably tetheredstructure reacts specifically are more than one epitope of the sameproinflammatory effector of the innate immune system or more than oneepitope of the same coagulation factor.

Thus, “two different targets” can refer to two different antigens, or totwo different epitopes of the same antigen. Multiple antibodies may beused against the same antigen, thus increasing valency. For example,when targeting MIF or HMGB-1, particularly for the treatment of sepsis,some cancers, and atherosclerotic plaques, two antibodies binding to twoidentical epitopes of the targets can be incorporated into a stablytethered structure with another antibody having one or more binding armsto a different antigen, such as an HLA class II invariant chain antigen,such as CD74. The antibodies may be selected to bind to two differentantigens, e.g., antibodies to MIF and CD74; antibodies to HMGB-1 andCD74.

When a proinflammatory effector receptor is targeted, in a preferredembodiment the actual target may be an extracellular domain of theproinflammatory effector receptor. In an alternative embodiment, thestably tethered structure may comprise at least one molecule reactivewith a proinflammatory effector receptor. This molecule may be a naturalantagonist for said proinflammatory effector receptor, or a fragment ormutant of this antagonist that interacts specifically with the receptor.In a preferred embodiment, the natural antagonist is the natural IL-1receptor antagonist, or a fragment or mutant of this antagonist.

In one embodiment, a target may be an antigen or receptor of theadaptive immune system. In other embodiments, the target of the stablytethered structure may occur on cells of the innate immune system, suchas granulocytes, monocytes, macrophages, dendritic cells, and NK-cells.Other targets include platelets and endothelial cells. Yet another groupof targets is the group consisting of C5a, LPS, IFNγ and B7. A furthergroup of suitable targets include CD2, CD3, CD4, CD14, CD18, CD11a,CD20, CD22, CD23, CD25, CD29, CD38, CD40L, CD52, CD64, CD83, CD147, andCD154. The CDs are targets on immune cells, which can be blocked toprevent an immune cell response. CD83 is particularly useful as a markerof activated dendritic cells (Cao et al., Biochem J., Aug. 23, 2004(Epub ahead of print); Zinser et al., J. Exp Med. 200(3):345-51 (2004)).

Certain targets are of particular interest, such as MIF, HMGB-1, TNF-α,the complement factors and complement regulatory proteins, and thecoagulation factors. MIF is a pivotal cytokine of the innate immunesystem and plays an important part in the control of inflammatoryresponses. Originally described as a T lymphocyte-derived factor thatinhibited the random migration of macrophages, the protein known asmacrophage migration inhibitory factor (MIF) was an enigmatic cytokinefor almost 3 decades. In recent years, the discovery of MIF as a productof the anterior pituitary gland and the cloning and expression ofbioactive, recombinant MIF protein have led to the definition of itscritical biological role in vivo. MIF has the unique property of beingreleased from macrophages and T lymphocytes that have been stimulated byglucocorticoids. Once released, MIF overcomes the inhibitory effects ofglucocorticoids on TNF-α, IL-1 beta, IL-6, and IL-8 production byLPS-stimulated monocytes in vitro and suppresses the protective effectsof steroids against lethal endotoxemia in vivo. MIF also antagonizesglucocorticoid inhibition of T-cell proliferation in vitro by restoringIL-2 and IFN-gamma production. MIF is the first mediator to beidentified that can counter-regulate the inhibitory effects ofglucocorticoids and thus plays a critical role in the host control ofinflammation and immunity. MIF is particularly useful in treatingcancer, pathological angiogenesis, and sepsis or septic shock.

HMGB-1, a DNA binding nuclear and cytosolic protein, is aproinflammatory cytokine released by monocytes and macrophages that havebeen activated by IL-1β, TNF, or LPS. Via its B box domain, it inducesphenotypic maturation of DCs. It also causes increased secretion of theproinflammatory cytokines IL-1 alpha, IL-6, IL-8, IL-12, TNF-α andRANTES. HMGB-1 released by necrotic cells may be a signal of tissue orcellular injury that, when sensed by DCs, induces and/or enhances animmune reaction. Palumbo et al. report that HMBG1 induces mesoangioblastmigration and proliferation (J Cell Biol, 164:441-449 (2004)).

HMGB-1 is a late mediator of endotoxin-induced lethality that exhibitssignificantly delayed kinetics relate to TNF and IL-1beta. Experimentaltherapeutics that target specific early inflammatory mediators such asTNF and IL-1beta alone have not proven efficacious in the clinic, butstably tethered structures can improve response by targeting both earlyand late inflammatory inflammatory mediators.

Stably tethered structures that target HMBG-1 are especially useful intreating arthritis, particularly collagen-induced arthritis. Stablytethered structures comprising HMBG-1 also are useful in treating sepsisand/or septic shock. Yang et al., PNAS USA 101:296-301 (2004); Kokkolaet al., Arthritis Rheum, 48:2052-8 (2003); Czura et al., J Infect Dis,187 Suppl 2:S391-6 (2003); Treutiger et al., J Intern Med, 254:375-85(2003).

TNF-α is an important cytokine involved in systemic inflammation and theacute phase response. TNF-α is released by stimulated monocytes,fibroblasts, and endothelial cells. Macrophages, T-cells andB-lymphocytes, granulocytes, smooth muscle cells, eosinophils,chondrocytes, osteoblasts, mast cells, glial cells, and keratinocytesalso produce TNF-α after stimulation. Its release is stimulated byseveral other mediators, such as interleukin-1 and bacterial endotoxin,in the course of damage, e.g., by infection. It has a number of actionson various organ systems, generally together with interleukins-1 and -6.One of the actions of TNF-α is appetite suppression; hence stablytethered structures for treating cachexia preferably target TNF-α. Italso stimulates the acute phase response of the liver, leading to anincrease in C-reactive protein and a number of other mediators. It alsois a useful target when treating sepsis or septic shock.

The complement system is a complex cascade involving proteolyticcleavage of serum glycoproteins often activated by cell receptors. The“complement cascade” is constitutive and non-specific but it must beactivated in order to function. Complement activation results in aunidirectional sequence of enzymatic and biochemical reactions. In thiscascade, a specific complement protein, C5, forms two highly active,inflammatory byproducts, C5a and C5b, which jointly activate white bloodcells. This in turn evokes a number of other inflammatory byproducts,including injurious cytokines, inflammatory enzymes, and cell adhesionmolecules. Together, these byproducts can lead to the destruction oftissue seen in many inflammatory diseases. This cascade ultimatelyresults in induction of the inflammatory response, phagocyte chemotaxisand opsonization, and cell lysis.

The complement system can be activated via two distinct pathways, theclassical pathway and the alternate pathway. Most of the complementcomponents are numbered (e.g., C1, C2, C3, etc.) but some are referredto as “Factors.” Some of the components must be enzymatically cleaved toactivate their function; others simply combine to form complexes thatare active. Active components of the classical pathway include C1q, C1r,C1s, C2a, C2b, C3a, C3b, C4a, and C4b. Active components of thealternate pathway include C3a, C3b, Factor B, Factor Ba, Factor Bb,Factor D, and Properdin. The last stage of each pathway is the same, andinvolves component assembly into a membrane attack complex. Activecomponents of the membrane attack complex include C5a, C5b, C6, C7, C8,and C9n.

While any of these components of the complement system can be targetedby a stably tethered structure, certain of the complement components arepreferred. C3a, C4a and C5a cause mast cells to release chemotacticfactors such as histamine and serotonin, which attract phagocytes,antibodies and complement, etc. These form one group of preferredtargets. Another group of preferred targets includes C3b, C4b and C5b,which enhance phagocytosis of foreign cells. Another preferred group oftargets are the predecessor components for these two groups, i.e., C3,C4 and C5. C5b, C6, C7, C8 and C9 induce lysis of foreign cells(membrane attack complex) and form yet another preferred group oftargets.

Complement C5a, like C3a, is an anaphylatoxin. It mediates inflammationand is a chemotactic attractant for induction of neutrophilic release ofantimicrobial proteases and oxygen radicals. Therefore, C5a and itspredecessor C5 are particularly preferred targets. By targeting C5, notonly is C5a affected, but also C5b, which initiates assembly of themembrane-attack complex. Thus, C5 is another preferred target. C3b, andits predecessor C3, also are preferred targets, as both the classicaland alternate complement pathways depend upon C3b. Three proteins affectthe levels of this factor, C1 inhibitor, protein H and Factor I, andthese are also preferred targets according to the invention. Complementregulatory proteins, such as CD46, CD55, and CD59, may be targets towhich the stably tethered structures bind.

Coagulation factors also are preferred targets, particularly tissuefactor (TF) and thrombin. TF is also known also as tissuethromboplastin, CD142, coagulation factor III, or factor III. TF is anintegral membrane receptor glycoprotein and a member of the cytokinereceptor superfamily. The ligand binding extracellular domain of TFconsists of two structural modules with features that are consistentwith the classification of TF as a member of type-2 cytokine receptors.TF is involved in the blood coagulation protease cascade and initiatesboth the extrinsic and intrinsic blood coagulation cascades by forminghigh affinity complexes between the extracellular domain of TF and thecirculating blood coagulation factors, serine proteases factor VII orfactor VIIa. These enzymatically active complexes then activate factorIX and factor X, leading to thrombin generation and clot formation.

TF is expressed by various cell types, including monocytes, macrophagesand vascular endothelial cells, and is induced by IL-1, TNF-α orbacterial lipopolysaccharides. Protein kinase C is involved in cytokineactivation of endothelial cell TF expression. Induction of TF byendotoxin and cytokines is an important mechanism for initiation ofdisseminated intravascular coagulation seen in patients withGram-negative sepsis. TF also appears to be involved in a variety ofnon-hemostatic functions including inflammation, cancer, brain function,immune response, and tumor-associated angiogenesis. Thus, stablytethered structures that target TF are useful not only in the treatmentof coagulopathies, but also in the treatment of sepsis, cancer,pathologic angiogenesis, and other immune and inflammatory dysregulatorydiseases according to the invention. A complex interaction between thecoagulation pathway and the cytokine network is suggested by the abilityof several cytokines to influence TF expression in a variety of cellsand by the effects of ligand binding to the receptor. Ligand binding(factor VIIa) has been reported to give an intracellular calcium signal,thus indicating that TF is a true receptor.

Thrombin is the activated form of coagulation factor II (prothrombin);it converts fibrinogen to fibrin. Thrombin is a potent chemotaxin formacrophages, and can alter their production of cytokines and arachidonicacid metabolites. It is of particular importance in the coagulopathiesthat accompany sepsis. Numerous studies have documented the activationof the coagulation system either in septic patients or following LPSadministration in animal models. Despite more than thirty years ofresearch, the mechanisms of LPS-induced liver toxicity remain poorlyunderstood. It is now clear that they involve a complex and sequentialseries of interactions between cellular and humoral mediators. In thesame period of time, gram-negative systemic sepsis and its sequallaehave become a major health concern, attempts to use monoclonalantibodies directed against LPS or various inflammatory mediators haveyielded only therapeutic failures. Stably tethered structures thattarget both thrombin and at least one other target address the clinicalfailures in sepsis treatment.

In other embodiments, the stably tethered structures bind to a MHC classI, MHC class II or accessory molecule, such as CD40, CD54, CD80 or CD86.The stably tethered structure also may bind to a T-cell activationcytokine, or to a cytokine mediator, such as NF-κB.

In certain embodiments, one of the two different targets may be a cancercell receptor or cancer-associated antigen, particularly one that isselected from the group consisting of B-cell lineage antigens (CD19,CD20, CD21, CD22, CD23, etc.), VEGFR, EGFR, carcinoembryonic antigen(CEA), placental growth factor (P1GF), tenascin, HER-2/neu, EGP-1,EGP-2, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD74, CD80, CD138,NCA66, CEACAM6 (carcinoembryonic antigen-related cellular adhesionmolecule 6), MUC1, MUC2, MUC3, MUC4, MUC16, IL-6, α-fetoprotein (AFP),A3, CA125, colon-specific antigen-p (CSAp), folate receptor, HLA-DR,human chorionic gonadotropin (HCG), Ia, EL-2, insulin-like growth factor(ILGF) and ILGF receptor, KS-1, Le(y), MAGE, necrosis antigens, PAM-4,prostatic acid phosphatase (PAP), Pr1, prostate specific antigen (PSA),prostate specific membrane antigen (PSMA), 5100, T101, TAC, TAG72, TRAILreceptors, and carbonic anhydrase IX.

Targets associated with sepsis and immune dysregulation and other immunedisorders include MIF, IL-1, IL-6, IL-8, CD74, CD83, and C5aR.Antibodies and inhibitors against C5aR have been found to improvesurvival in rodents with sepsis (Huber-Lang et al., FASEB J 2002;16:1567-1574; Riedemann et al., J Clin Invest 2002; 110:101-108) andseptic shock and adult respiratory distress syndrome in monkeys (Hangenet al., J Surg Res 1989; 46:195-199; Stevens et al., J Clin Invest 1986;77:1812-1816). Thus, for sepsis, one of the two different targetspreferably is a target that is associated with infection, such asLPS/C5a. Other preferred targets include HMGB-1, TF, CD14, VEGF, andIL-6, each of which is associated with septicemia or septic shock.Preferred stably tethered structures are those that target two or moretargets from HMGB-1, TF and MIF, such as MIF/TF, and HMGB-1/TF.

In still other embodiments, one of the two different targets may be atarget that is associated with graft versus host disease or transplantrejection, such as MIF (Lo et al., Bone Marrow Transplant, 30(6):375-80(2002)). One of the two different targets also may be one thatassociated with acute respiratory distress syndrome, such as IL-8(Bouros et al., PMC Pulm Med, 4(1):6 (2004), atherosclerosis orrestenosis, such as MIF (Chen et al., Arterioscler Thromb Vasc Biol,24(4):709-14 (2004), asthma, such as IL-18 (Hata et al., Int Immunol,Oct. 11, 2004 Epub ahead of print), a granulomatous disease, such asTNF-α (Ulbricht et al., Arthritis Rheum, 50(8):2717-8 (2004), aneuropathy, such as carbamylated EPO (erythropoietin) (Leist et al.,Science 305(5681):164-5 (2004), or cachexia, such as IL-6 and TNF-α.

Other targets include C5a, LPS, IFN-gamma, B7; CD2, CD4, CD14, CD18,CD11a, CD11b, CD11c, CD14, CD18, CD27, CD29, CD38, CD40L, CD52, CD64,CD83, CD147, CD154. Activation of mononuclear cells by certain microbialantigens, including LPS, can be inhibited to some extent by antibodiesto CD18, CD11b, or CD11c, which thus implicate β₂-integrins (Cuzzola etal., J Immunol 2000; 164:5871-5876; Medvedev et al., J Immunol 1998;160: 4535-4542). CD83 has been found to play a role in giant cellarteritis (GCA), which is a systemic vasculitis that affects medium- andlarge-size arteries, predominately the extracranial branches of theaortic arch and of the aorta itself, resulting in vascular stenosis andsubsequent tissue ischemia, and the severe complications of blindness,stroke and aortic arch syndrome (Weyand and Goronzy, N Engl J Med 2003;349:160-169; Hunder and Valente, In: Inflammatory Diseases of BloodVessels. G. S. Hoffman and C. M. Weyand, eds, Marcel Dekker, New York,2002; 255-265). Antibodies to CD83 were found to abrogate vasculitis ina SCID mouse model of human GCA (Ma-Krupa et al., J Exp Med 2004;199:173-183), suggesting to these investigators that dendritic cells,which express CD83 when activated, are critical antigen-processing cellsin GCA. In these studies, they used a mouse anti-CD83 Mab (IgG1 cloneHB15e from Research Diagnostics). CD154, a member of the TNF family, isexpressed on the surface of CD4-positive T-lymphocytes, and it has beenreported that a humanized monoclonal antibody to CD154 producedsignificant clinical benefit in patients with active systemic lupuserythematosus (SLE) (Grammar et al., J Clin Invest 2003; 112:1506-1520).It also suggests that this antibody might be useful in other autoimmunediseases (Kelsoe, J Clin Invest 2003; 112:1480-1482). Indeed, thisantibody was also reported as effective in patients with refractoryimmune thrombocytopenic purpura (Kuwana et al., Blood 2004;103:1229-1236).

In rheumatoid arthritis, a recombinant interleukin-1 receptorantagonist, IL-1Ra or anakinra (Kineret®), has shown activity (Cohen etal., Ann Rheum Dis 2004; 63:1062-8; Cohen, Rheum Dis Clin North Am 2004;30:365-80). An improvement in treatment of these patients, whichhitherto required concomitant treatment with methotrexate, is to combineanakinra with one or more of the anti-proinflammatory effector cytokinesor anti-proinflammatory effector chemokines (as listed above). Indeed,in a review of antibody therapy for rheumatoid arthritis, Taylor (CurrOpin Pharmacol 2003; 3:323-328) suggests that in addition to TNF, otherantibodies to such cytokines as IL-1, IL-6, IL-8, IL-15, IL-17 andIL-18, are useful.

Some of the more preferred target combinations include the following.This is a list of examples of preferred combinations, but is notintended to be exhaustive.

First target Second target MIF A second proinflammatory effectorcytokine, especially HMGB-1, TNF-α, IL-1, or IL-6 MIF Proinflammatoryeffector chemokine, especially MCP-1, RANTES, MIP- 1A, or MIP-1B MIFProinflammatory effector receptor, especially IL-6R IL-13R, and IL-15RMIF Coagulation factor, especially TF or thrombin MIF Complement factor,especially C3, C5, C3a, or C5a MIF Complement regulatory protein,especially CD46, CD55, CD59, and mCRP MIF Cancer associated antigen orreceptor HMGB-1 A second proinflammatory effector cytokine, especiallyMIF, TNF-α, IL-1, or IL-6 HMGB-1 Proinflammatory effector chemokine,especially MCP-1, RANTES, MIP- 1A, or MIP-1B HMGB-1 Proinflammatoryeffector receptor especially MCP-1, RANTES, MIP-1A, or MIP-1B HMGB-1Coagulation factor, especially TF or thrombin HMGB-1 Complement factor,especially C3, C5, C3a, or C5a HMGB-1 Complement regulatory protein,especially CD46, CD55, CD59, and mCRP HMGB-1 Cancer associated antigenor receptor TNF-α A second proinflammatory effector cytokine, especiallyMIF, HMGB-1, TNF-α, IL-1, or IL-6 TNF-α Proinflammatory effectorchemokine, especially MCP-1, RANTES, MIP- 1A, or MIP-1B TNF-αProinflammatory effector receptor, especially IL-6R IL-13R, and IL-15RTNF-α Coagulation factor, especially TF or thrombin TNF-α Complementfactor, especially C3, C5, C3a, or C5a TNF-α Complement regulatoryprotein, especially CD46, CD55, CD59, and mCRP TNF-α Cancer associatedantigen or receptor LPS Proinflammatory effector cytokine, especiallyMIF, HMGB-1, TNF-α, IL-1, or IL-6 LPS Proinflammatory effectorchemokine, especially MCP-1, RANTES, MIP- 1A, or MIP-1B LPSProinflammatory effector receptor, especially IL-6R IL-13R, and IL-15RLPS Coagulation factor, especially TF or thrombin LPS Complement factor,especially C3, C5, C3a, or C5a LPS Complement regulatory protein,especially CD46, CD55, CD59, and mCRP TF or thrombin Proinflammatoryeffector cytokine, especially MIF, HMGB-1, TNF-α, IL-1, or IL-6 TF orthrombin Proinflammatory effector chemokine, especially MCP-1, RANTES,MIP- 1A, or MIP-1B TF or thrombin Proinflammatory effector receptor,especially IL-6R IL-13R, and IL-15R TF or thrombin Complement factor,especially C3, C5, C3a, or C5a TF or thrombin Complement regulatoryprotein, especially CD46, CD55, CD59, and mCRP TF or thrombin Cancerassociated antigen or receptor

The stably tethered structure may be a mixture that contains at leasttwo separate antibodies and/or receptors or their ligands that bind tothe different targets. In one preferred embodiment the targets areselected from the group consisting of proinflammatory effectors of theinnate immune system, coagulation factors, complement factors andcomplement regulatory proteins, and targets specifically associated withan inflammatory or immune-dysregulatory disorder, with a pathologicangiogenesis or cancer, or with an infectious disease.

The stably tethered structure may bind to a receptor or to its targetmolecule, such as for LPS, IL-1, IL-10, IL-6, MIF, HMGB1, TNF, IFN,tissue factor, thrombin, CD14, CD27, and CD134. Many of these exist asboth receptors and as soluble forms in the blood. Binding by the stablytethered structure results in rapid clearance from the blood, and thentargeting by the second component of the stably tethered structure toanother cell, such as a macrophage, for transport and degradation by thecell, especially the lysosomes. This is particularly effective when thesecond targeting component is against an internalizing antigen, such asCD74, expressed by macrophages and dendritic cells. This is consistentwith the disclosure of Hansen, U.S. Pat. No. 6,458,933, but focusing oninflammatory cytokines and other immune modulation molecules andreceptors for immune-dysregulation diseases, and cancer antigens for theimmunotherapy of these cancers.

Preferred stably tethered structures for the treatment of cancer includeantibodies to CD55 and to any of the above cancer antigens, antibodiesto CD46 and to any of the above cancer antigens, antibodies to CD59 andto any of the above cancer antigens, antibodies to MIF and to any of theabove cancer antigens, antibodies to NF-KB and any of the above cancerantigens, and antibodies to IL-6 and to any of the above cancer antigensother than IL-6.

The stably tethered structure may be used in conjunction with one ormore secondary therapeutics. This secondary therapeutic may be one thataffects a component of the innate immune system. Alternatively, it mayaffect a component of the adaptive immune system. The secondarytherapeutic may also be a component that affects coagulation, cancer, oran autoimmune disease, such as a cytotoxic drug.

The stably tethered structure with a diagnostic or therapeutic agent maybe provided as a kit for human or mammalian therapeutic and diagnosticuse in a pharmaceutically acceptable injection vehicle, preferablyphosphate-buffered saline (PBS) at physiological pH and concentration.The preparation preferably will be sterile, especially if it is intendedfor use in humans. Optional components of such kits include stabilizers,buffers, labeling reagents, radioisotopes, paramagnetic compounds,second antibody for enhanced clearance, and conventional syringes,columns, vials and the like.

Phage Display

In some alternative embodiments, binding peptides for construction ofDDD and/or AD domains may be determined by phage display methods thatare well known in the art. For example, peptides that bind to DDDdomains and that therefore may be substituted for naturally occurring ADsequences may be identified by phage display panning against a DDD dimerand selecting for phage with high binding affinity. Other types ofbinding peptides that are selective or specific for particular targetmolecules may be detected by phage display panning against the selectedtarget.

Various methods of phage display and techniques for producing diversepopulations of peptides are well known in the art. For example, U.S.Pat. Nos. 5,223,409; 5,622,699 and 6,068,829, each of which isincorporated herein by reference, disclose methods for preparing a phagelibrary. The phage display technique involves genetically manipulatingbacteriophage so that small peptides can be expressed on their surface(Smith and Scott, 1985, Science 228:1315-1317; Smith and Scott, 1993,Meth. Enzymol. 21:228-257).

The past decade has seen considerable progress in the construction ofphage-displayed peptide libraries and in the development of screeningmethods in which the libraries are used to isolate peptide ligands. Forexample, the use of peptide libraries has made it possible tocharacterize interacting sites and receptor-ligand binding motifs withinmany proteins, such as antibodies involved in inflammatory reactions orintegrins that mediate cellular adherence. This method has also beenused to identify novel peptide ligands that may serve as leads to thedevelopment of peptidomimetic drugs or imaging agents (Arap et al.,1998a, Science 279:377-380). In addition to peptides, larger proteindomains such as single-chain antibodies may also be displayed on thesurface of phage particles (Arap et al., 1998a).

Targeting amino acid sequences selective for a given target molecule maybe isolated by panning (Pasqualini and Ruoslahti, 1996, Nature380:364-366; Pasqualini, 1999, The Quart. J. Nucl. Med. 43:159-162). Inbrief, a library of phage containing putative targeting peptides isadministered to target molecules and samples containing bound phage arecollected. Target molecules may, for example, be attached to the bottomof microtiter wells in a 96-well plate. Phage that bind to a target maybe eluted and then amplified by growing them in host bacteria.

In certain embodiments, the phage may be propagated in host bacteriabetween rounds of panning. Rather than being lysed by the phage, thebacteria may instead secrete multiple copies of phage that display aparticular insert. If desired, the amplified phage may be exposed to thetarget molecule again and collected for additional rounds of panning.Multiple rounds of panning may be performed until a population ofselective or specific binders is obtained. The amino acid sequence ofthe peptides may be determined by sequencing the DNA corresponding tothe targeting peptide insert in the phage genome. The identifiedtargeting peptide may then be produced as a synthetic peptide bystandard protein chemistry techniques (Arap et al., 1998a, Smith et al.,1985).

Aptamers

In certain embodiments, a precursor for construct formation may comprisean aptamer. Methods of constructing and determining the bindingcharacteristics of aptamers are well known in the art. For example, suchtechniques are described in U.S. Pat. Nos. 5,582,981, 5,595,877 and5,637,459, each incorporated herein by reference.

Aptamers may be prepared by any known method, including synthetic,recombinant, and purification methods, and may be used alone or incombination with other ligands specific for the same target. In general,a minimum of approximately 3 nucleotides, preferably at least 5nucleotides, are necessary to effect specific binding. Aptamers ofsequences shorter than 10 bases may be feasible, although aptamers of10, 20, 30 or 40 nucleotides may be preferred.

Aptamers need to contain the sequence that confers binding specificity,but may be extended with flanking regions and otherwise derivatized. Inpreferred embodiments, the binding sequences of aptamers may be flankedby primer-binding sequences, facilitating the amplification of theaptamers by PCR or other amplification techniques. In a furtherembodiment, the flanking sequence may comprise a specific sequence thatpreferentially recognizes or binds a moiety to enhance theimmobilization of the aptamer to a substrate.

Aptamers may be isolated, sequenced, and/or amplified or synthesized asconventional DNA or RNA molecules. Alternatively, aptamers of interestmay comprise modified oligomers. Any of the hydroxyl groups ordinarilypresent in aptamers may be replaced by phosphonate groups, phosphategroups, protected by a standard protecting group, or activated toprepare additional linkages to other nucleotides, or may be conjugatedto solid supports. One or more phosphodiester linkages may be replacedby alternative linking groups, such as P(O)O replaced by P(O)S, P(O)NR₂,P(O)R, P(O)OR′, CO, or CNR₂, wherein R is H or alkyl (1-20C) and R′ isalkyl (1-20C); in addition, this group may be attached to adjacentnucleotides through O or S, Not all linkages in an oligomer need to beidentical.

Methods for preparation and screening of aptamers that bind toparticular targets of interest are well known, for example U.S. Pat. No.5,475,096 and U.S. Pat. No. 5,270,163, each incorporated by reference.The technique generally involves selection from a mixture of candidateaptamers and step-wise iterations of binding, separation of bound fromunbound aptamers and amplification. Because only a small number ofsequences (possibly only one molecule of aptamer) corresponding to thehighest affinity aptamers exist in the mixture, it is generallydesirable to set the partitioning criteria so that a significant amountof aptamers in the mixture (approximately 5-50%) is retained duringseparation. Each cycle results in an enrichment of aptamers with highaffinity for the target. Repetition for between three to six selectionand amplification cycles may be used to generate aptamers that bind withhigh affinity and specificity to the target.

Avimers

In certain embodiments, the precursors, components and/or complexesdescribed herein may comprise one or more avimer sequences. Avimers area class of binding proteins somewhat similar to antibodies in theiraffinities and specifities for various target molecules. They weredeveloped from human extracellular receptor domains by in vitro exonshuffling and phage display. (Silverman et al., 2005, Nat. Biotechnol.23:1493-94; Silverman et al., 2006, Nat. Biotechnol. 24:220.) Theresulting multidomain proteins may comprise multiple independent bindingdomains, that may exhibit improved affinity (in some casessub-nanomolar) and specificity compared with single-epitope bindingproteins. (Id.) In various embodiments, avimers may be attached to, forexample, AD and/or DDD sequences for use in the claimed methods andcompositions. Additional details concerning methods of construction anduse of avimers are disclosed, for example, in U.S. Patent ApplicationPublication Nos. 20040175756, 20050048512, 20050053973, 20050089932 and20050221384, the Examples section of each of which is incorporatedherein by reference.

Proteins and Peptides

A variety of polypeptides or proteins may be used within the scope ofthe claimed methods and compositions. In certain embodiments, theproteins may comprise antibodies or fragments of antibodies containingan antigen-binding site. In other embodiments, a protein or peptide maybe an effector molecule, such as an enzyme, hormone, cytokine, bindingprotein or toxin.

As used herein, a protein, polypeptide or peptide generally refers, butis not limited to, a protein of greater than about 200 amino acids, upto a full length sequence translated from a gene; a polypeptide ofgreater than about 100 amino acids; and/or a peptide of from about 3 toabout 100 amino acids. For convenience, the terms “protein,”“polypeptide” and “peptide” are used interchangeably herein.Accordingly, the term “protein or peptide” encompasses amino acidsequences comprising at least one of the 20 common amino acids found innaturally occurring proteins, or at least one modified or unusual aminoacid.

As used herein, an “amino acid residue” refers to any naturallyoccurring amino acid, any amino acid derivative or any amino acid mimicknown in the art. In certain embodiments, the residues of the protein orpeptide are sequential, without any non-amino acid interrupting thesequence of amino acid residues. In other embodiments, the sequence maycomprise one or more non-amino acid moieties. In particular embodiments,the sequence of residues of the protein or peptide may be interrupted byone or more non-amino acid moieties.

Accordingly, the term “protein or peptide” encompasses amino acidsequences comprising at least one of the 20 common amino acids found innaturally occurring proteins, or at least one modified or unusual aminoacid, including but not limited to: 2-Aminoadipic acid, 3-Aminoadipicacid, β-alanine, β-Amino-propionic acid, 2-Aminobutyric acid,4-Aminobutyric acid, piperidinic acid, 6-Aminocaproic acid,2-Aminoheptanoic acid, 2-Aminoisobutyric acid, 3-Aminoisobutyric acid,2-Aminopimelic acid, 2,4-Diaminobutyric acid, Desmosine,2,2′-Diaminopimelic acid, 2,3-Diaminopropionic acid, N-Ethylasparagine,Hydroxylysine, allo-Hydroxylysine, 3-Hydroxyproline, 4-Hydroxyproline,Isodesmosine, allo-Isoleucine, N-Methylglycine, sarcosine,N-Methylisoleucine, 6-N-Methyllysine, N-Methylvaline, NorvalineNorleucine and Ornithine. Alternatively, proteins or peptides maycomprise one or more D-amino acids in addition to or instead of thenaturally occurring L-amino acids. Methods of producing peptidesincorporating D-amino acids are disclosed, for example, in U.S. PatentApplication Publication No. 20050025709, McBride et al., filed Jun. 14,2004.

Proteins or peptides may be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, or the chemical synthesisof proteins or peptides. The nucleotide and protein, polypeptide andpeptide sequences corresponding to various genes have been previouslydisclosed and may be found at computerized databases known to those ofordinary skill in the art. One such database is the National Center forBiotechnology Information's Genbank and GenPept databases. The codingregions for known genes may be amplified and/or expressed using thetechniques disclosed herein or as would be know to those of ordinaryskill in the art. Alternatively, various commercial preparations ofproteins, polypeptides, and peptides are known to those of skill in theart.

Peptide Mimetics

Another embodiment for the preparation of polypeptides is the use ofpeptide mimetics. Mimetics are peptide-containing molecules that mimicelements of protein secondary structure. See, for example, Johnson etal., “Peptide Turn Mimetics” in BIOTECHNOLOGY AND PHARMACY, Pezzuto etal., Eds., Chapman and Hall, New York (1993), incorporated herein byreference. The rationale behind the use of peptide mimetics is that thepeptide backbone of proteins exists chiefly to orient amino acid sidechains so as to facilitate molecular interactions, such as those ofantibody and antigen. A peptide mimetic is expected to permit molecularinteractions similar to the natural molecule. These principles may beused to engineer second generation molecules having many of the naturalproperties of the binding peptides disclosed herein, but with altered orimproved characteristics, such as increased absorption across thestomach or intestine and/or improved stability or activity in vivo.

Fusion Proteins

Various embodiments may concern fusion proteins. These moleculesgenerally have all or a substantial portion of a peptide, linked at theN- or C-terminus, to all or a portion of a second polypeptide orprotein. For example, fusions may employ leader sequences from otherspecies to permit the recombinant expression of a protein in aheterologous host. Another useful fusion includes the addition of animmunologically active domain, such as an antibody epitope. Yet anotheruseful form of fusion may include attachment of a moiety of use forpurification, such as the FLAG epitope (Prickett et al., 1989,Biotechniques 7:580-589; Castrucci et al., 1992, J Virol 66:4647-4653).Another use of fusion proteins would concern construction of componentsof the tethered complexes claimed herein, for example to provide a DDDsequence attached to a first monomer and an AD sequence attached to asecond monomer.

Methods of generating fusion proteins are well known to those of skillin the art. Such proteins may be produced, for example, by chemicalattachment using bifunctional cross-linking reagents, by de novosynthesis of the complete fusion protein, or by attachment of a DNAsequence encoding a first protein or peptide to a DNA sequence encodinga second peptide or protein, followed by expression of the intact fusionprotein, as exemplified in the following Examples.

Synthetic Peptides

Proteins or peptides may be synthesized, in whole or in part, insolution or on a solid support in accordance with conventionaltechniques. Various automatic synthesizers are commercially availableand can be used in accordance with known protocols. See, for example,Stewart and Young, (1984, Solid Phase Peptide Synthesis, 2d. ed., PierceChemical Co.); Tam et al., (1983, J. Am. Chem. Soc., 105:6442);Merrifield, (1986, Science, 232: 341-347); and Barany and Merrifield(1979, The Peptides, Gross and Meienhofer, eds., Academic Press, NewYork, pp. 1-284). Short peptide sequences, usually from about 6 up toabout 35 to 50 amino acids, can be readily synthesized by such methods.Alternatively, recombinant DNA technology may be employed wherein anucleotide sequence which encodes a peptide of interest is inserted intoan expression vector, transformed or transfected into an appropriatehost cell, and cultivated under conditions suitable for expression.

Peptide Administration

Various embodiments of the claimed methods and/or compositions mayconcern one or more peptide based stably tethered structures to beadministered to a subject. Administration may occur by any route knownin the art, including but not limited to oral, nasal, buccal,inhalational, rectal, vaginal, topical, orthotopic, intradermal,subcutaneous, intramuscular, intraperitoneal, intraarterial, intrathecalor intravenous injection.

Unmodified peptides administered orally to a subject can be degraded inthe digestive tract and depending on sequence and structure may exhibitpoor absorption across the intestinal lining. However, methods forchemically modifying peptides to render them less susceptible todegradation by endogenous proteases or more absorbable through thealimentary tract are well known (see, for example, Blondelle et al.,1995, Biophys. J. 69:604-11; Ecker and Crooke, 1995, Biotechnology13:351-69; Goodman and Ro, 1995, BURGER'S MEDICINAL CHEMISTRY AND DRUGDISCOVERY, VOL. I, ed. Wollf, John Wiley & Sons; Goodman and Shao, 1996,Pure & Appl. Chem. 68:1303-08). Methods for preparing libraries ofpeptide analogs, such as peptides containing D-amino acids;peptidomimetics consisting of organic molecules that mimic the structureof a peptide; or peptoids such as vinylogous peptoids, have also beendescribed and may be used to construct peptide based stably tetheredstructures suitable for oral administration to a subject.

In certain embodiments, the standard peptide bond linkage may bereplaced by one or more alternative linking groups, such as CH₂—NH,CH₂—S, CH₂—CH₂, CH═CH, CO—CH₂, CHOH—CH₂ and the like. Methods forpreparing peptide mimetics are well known (for example, Hruby, 1982,Life Sci 31:189-99; Holladay et al., 1983, Tetrahedron Lett. 24:4401-04;Jennings-White et al., 1982, Tetrahedron Lett. 23:2533; Almquiest etal., 1980, J. Med. Chem. 23:1392-98; Hudson et al., 1979, Int. J. Pept.Res. 14:177-185; Spatola et al., 1986, Life Sci 38:1243-49; U.S. Pat.Nos. 5,169,862; 5,539,085; 5,576,423, 5,051,448, 5,559,103, eachincorporated herein by reference.) Peptide mimetics may exhibit enhancedstability and/or absorption in vivo compared to their peptide analogs.

Alternatively, peptides may be administered by oral delivery usingN-terminal and/or C-terminal capping to prevent exopeptidase activity.For example, the C-terminus may be capped using amide peptides and theN-terminus may be capped by acetylation of the peptide. Peptides mayalso be cyclized to block exopeptidases, for example by formation ofcyclic amides, disulfides, ethers, sulfides and the like.

Peptide stabilization may also occur by substitution of D-amino acidsfor naturally occurring L-amino acids, particularly at locations whereendopeptidases are known to act. Endopeptidase binding and cleavagesequences are known in the art and methods for making and using peptidesincorporating D-amino acids have been described (e.g., U.S. PatentApplication Publication No. 20050025709, McBride et al., filed Jun. 14,2004, incorporated herein by reference). In certain embodiments,peptides and/or proteins may be orally administered by co-formulationwith proteinase- and/or peptidase-inhibitors.

Other methods for oral delivery of therapeutic peptides are disclosed inMehta (“Oral delivery and recombinant production of peptide hormones,”June 2004, BioPharm International). The peptides are administered in anenteric-coated solid dosage form with excipients that modulateintestinal proteolytic activity and enhance peptide transport across theintestinal wall. Relative bioavailability of intact peptides using thistechnique ranged from 1% to 10% of the administered dosage. Insulin hasbeen successfully administered in dogs using enteric-coatedmicrocapsules with sodium cholate and a protease inhibitor (Ziv et al.,1994, J. Bone Miner. Res. 18 (Suppl. 2):792-94. Oral administration ofpeptides has been performed using acylcarnitine as a permeation enhancerand an enteric coating (Eudragit L30D-55, Rohm Pharma Polymers, seeMehta, 2004). Excipients of use for orally administered peptides maygenerally include one or more inhibitors of intestinalproteases/peptidases along with detergents or other agents to improvesolubility or absorption of the peptide, which may be packaged within anenteric-coated capsule or tablet (Mehta, 2004). Organic acids may beincluded in the capsule to acidify the intestine and inhibit intestinalprotease activity once the capsule dissolves in the intestine (Mehta,2004). Another alternative for oral delivery of peptides would includeconjugation to polyethylene glycol (PEG)-based amphiphilic oligomers,increasing absorption and resistance to enzymatic degradation (Solteroand Ekwuribe, 2001, Pharm. Technol. 6:110).

In still other embodiments, peptides may be modified for oral orinhalational administration by conjugation to certain proteins, such asthe Fc region of IgG1 (see Examples 3-7). Methods for preparation anduse of peptide-Fc conjugates are disclosed, for example, in Low et al.(2005, Hum. Reprod. 20:1805-13) and Dumont et al. (2005, J. Aerosol.Med. 18:294-303), each incorporated herein by reference. Low et al.(2005) disclose the conjugation of the alpha and beta subunits of FSH tothe Fc region of IgG1 in single chain or heterodimer form, usingrecombinant expression in CHO cells. The Fc conjugated peptides wereabsorbed through epithelial cells in the lung or intestine by theneonatal Fc receptor mediated transport system. The Fc conjugatedpeptides exhibited improved stability and absorption in vivo compared tothe native peptides. It was also observed that the heterodimer conjugatewas more active than the single chain form.

Cross-Linkers

In some embodiments, proteins, peptides or other macro-molecules may becovalently cross-linked using various cross-linking reagents known inthe art, such as homo-bifunctional, hetero-bifunctional and/orphotoactivatable cross-linking reagents. Non-limiting examples of suchreagents include bisimidates; 1,5-difluoro-2,4-(dinitrobenzene);N-hydroxysuccinimide ester of suberic acid; disuccinimidyl tartarate;dimethyl-3,3′-dithio-bispropionimidate;N-succinimidyl-3-(2-pyridyldithio)propionate;4-(bromoaminoethyl)-2-nitrophenylazide; and 4-azidoglyoxal. In anexemplary embodiment, a carbodiimide cross-linker, such as DCCD or EDC,may be used to cross-link acidic residues to amino or other groups. Suchreagents may be modified to attach various types of labels, such asfluorescent labels.

Bifunctional cross-linking reagents have been extensively used for avariety of purposes. Homobifunctional reagents that carry two identicalfunctional groups proved to be highly efficient in inducingcross-linking between identical and different macromolecules or subunitsof a macromolecule, and linking of polypeptide ligands to their specificbinding sites. Heterobifunctional reagents contain two differentfunctional groups. By taking advantage of the differential reactivitiesof the two different functional groups, cross-linking can be controlledboth selectively and sequentially. The bifunctional cross-linkingreagents can be divided according to the specificity of their functionalgroups, e.g., amino, sulfhydryl, guanidino, indole, carboxyl specificgroups. Of these, reagents directed to free amino groups have becomeespecially popular because of their commercial availability, ease ofsynthesis and the mild reaction conditions under which they can beapplied. A majority of heterobifunctional cross-linking reagentscontains a primary amine-reactive group and a thiol-reactive group.

In another example, heterobifunctional cross-linking reagents andmethods of using the cross-linking reagents are described (U.S. Pat. No.5,889,155). The cross-linking reagents combine a nucleophilic hydrazideresidue with an electrophilic maleimide residue, allowing coupling inone example, of aldehydes to free thiols. The cross-linking reagent canbe modified to cross-link various functional groups.

Antibodies

Various embodiments may concern antibodies for a target. The term“antibody” is used herein to refer to any antibody-like molecule thathas an antigen binding region, and includes antibody fragments such asFab′, Fab, F(ab′)₂, single domain antibodies (DABs), Fv, scFv (singlechain Fv), and the like. Techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (See, e.g., Harlowe and Lane, 1988, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory). Antibodies of use may also becommercially obtained from a wide variety of known sources. For example,a variety of antibody secreting hybridoma lines are available from theAmerican Type Culture Collection (ATCC, Manassas, Va.).

Production of Antibody Fragments

Some embodiments of the claimed methods and/or compositions may concernantibody fragments. Such antibody fragments may be obtained by pepsin orpapain digestion of whole antibodies by conventional methods. Forexample, antibody fragments may be produced by enzymatic cleavage ofantibodies with pepsin to provide F(ab′)₂ fragments. This fragment maybe further cleaved using a thiol reducing agent and, optionally,followed by a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce Fab′ monovalent fragments.Alternatively, an enzymatic cleavage using papain n produces twomonovalent Fab fragments and an Fc fragment. Exemplary methods forproducing antibody fragments are disclosed in U.S. Pat. No. 4,036,945;U.S. Pat. No. 4,331,647; Nisonoff et al., 1960, Arch. Biochem. Biophys.,89:230; Porter, 1959, Biochem. J., 73:119; Edelman et al., 1967, METHODSIN ENZYMOLOGY, page 422 (Academic Press), and Coligan et al. (eds.),1991, CURRENT PROTOCOLS IN IMMUNOLOGY, (John Wiley & Sons).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments or other enzymatic, chemical or genetic techniques also may beused, so long as the fragments bind to the antigen that is recognized bythe intact antibody. For example, Fv fragments comprise an associationof V_(H) and V_(L) chains. This association can be noncovalent, asdescribed in Inbar et al., 1972, Proc. Nat'l. Acad. Sci. USA, 69:2659.Alternatively, the variable chains may be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde. SeeSandhu, 1992, Crit. Rev. Biotech., 12:437.

Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (sFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains, connected by an oligonucleotideslinker sequence. The structural gene is inserted into an expressionvector that is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingsFv's are well-known in the art. See Whitlow et al., 1991, Methods: ACompanion to Methods in Enzymology 2:97; Bird et al., 1988, Science,242:423; U.S. Pat. No. 4,946,778; Pack et al., 1993, Bio/Technology,11:1271, and Sandhu, 1992, Crit. Rev. Biotech., 12:437.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See Larrick et al., 1991, Methods:A Companion to Methods in Enzymology 2:106; Ritter et al. (eds.), 1995,MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION,pages 166-179 (Cambridge University Press); Birch et al., (eds.), 1995,MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, pages 137-185(Wiley-Liss, Inc.).

Chimeric and Humanized Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. Methods for constructingchimeric antibodies are well known in the art (e.g., Leung et al., 1994,Hybridoma 13:469).

A chimeric monoclonal antibody may be humanized by transferring themouse CDRs from the heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. The mouse framework regions (FR) in the chimeric monoclonalantibody are also replaced with human FR sequences. To preserve thestability and antigen specificity of the humanized monoclonal, one ormore human FR residues may be replaced by the mouse counterpartresidues. Humanized monoclonal antibodies may be used for therapeutictreatment of subjects. The affinity of humanized antibodies for a targetmay also be increased by selected modification of the CDR sequences(WO0029584A1). Techniques for production of humanized monoclonalantibodies are well known in the art. (See, e.g., Jones et al., 1986,Nature, 321:522; Riechmann et al., Nature, 1988, 332:323; Verhoeyen etal., 1988, Science, 239:1534; Carter et al., 1992, Proc. Nat'l Acad.Sci. USA, 89:4285; Sandhu, Crit. Rev. Biotech., 1992, 12:437; Tempest etal., 1991, Biotechnology 9:266; Singer et al., J. Immunol., 1993,150:2844.)

Other embodiments may concern non-human primate antibodies. Generaltechniques for raising therapeutically useful antibodies in baboons maybe found, for example, in Goldenberg et al., WO 91/11465 (1991), and inLosman et al., Int. J. Cancer 46: 310 (1990).

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50; each incorporated herein by reference). Such fully humanantibodies are expected to exhibit even fewer side effects than chimericor humanized antibodies and to function in vivo as essentiallyendogenous human antibodies. In certain embodiments, the claimed methodsand procedures may utilize human antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40, incorporated herein by reference). Human antibodies may begenerated from normal humans or from humans that exhibit a particulardisease state, such as cancer (Dantas-Barbosa et al., 2005). Theadvantage to constructing human antibodies from a diseased individual isthat the circulating antibody repertoire may be biased towardsantibodies against disease-associated antigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97,incorporated herein by reference). Library construction was performedaccording to Andris-Widhopf et al. (2000, In: Phage Display LaboratoryManual, Barbas et al. (eds), 1^(st) edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. pp. 9.1 to 9.22, incorporatedherein by reference). The final Fab fragments were digested withrestriction endonucleases and inserted into the bacteriophage genome tomake the phage display library. Such libraries may be screened bystandard phage display methods, as known in the art. The skilled artisanwill realize that this technique is exemplary only and any known methodfor making and screening human antibodies or antibody fragments by phagedisplay may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. A non-limiting example of such a system is theXenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23,incorporated herein by reference) from Abgenix (Fremont, Calif.). In theXenoMouse® and similar animals, the mouse antibody genes have beeninactivated and replaced by functional human antibody genes, while theremainder of the mouse immune system remains intact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Such human antibodies may be coupled to other molecules bychemical cross-linking or other known methodologies. Transgenicallyproduced human antibodies have been shown to have therapeutic potential,while retaining the pharmacokinetic properties of normal humanantibodies (Green et al., 1999). The skilled artisan will realize thatthe claimed compositions and methods are not limited to use of theXenoMouse® system but may utilize any transgenic animal that has beengenetically engineered to produce human antibodies.

Methods of Disease Tissue Detection, Diagnosis and Imaging

Protein-Based In Vitro Diagnosis

The present invention contemplates the use of stably tethered structuresto screen biological samples in vitro and/or in vivo for the presence ofthe disease-associated antigens. In exemplary immunoassays, a stablytethered structure comprising an antibody, fusion protein, or fragmentthereof may be utilized in liquid phase or bound to a solid-phasecarrier, as described below. In preferred embodiments, particularlythose involving in vivo administration, the antibody or fragment thereofis humanized. Also preferred, the antibody or fragment thereof is fullyhuman. Still more preferred, the fusion protein comprises a humanized orfully human antibody. The skilled artisan will realize that a widevariety of techniques are known for determining levels of expression ofa particular gene and any such known method, such as immunoassay,RT-PCR, mRNA purification and/or cDNA preparation followed byhybridization to a gene expression assay chip may be utilized todetermine levels of expression in individual subjects and/or tissues.Exemplary in vitro assays of use include RIA, ELISA, sandwich ELISA,Western blot, slot blot, dot blot, and the like. Although suchtechniques were developed using intact antibodies, stably tetheredstructures that incorporate antibodies, antibody fragments or otherbinding moieties may be used.

Stably tethered structures incorporating antibodies, fusion proteins,antibody fragments and/or other binding moieties may also be used todetect the presence of a target antigen in tissue sections prepared froma histological specimen. Such in situ detection can be used to determinethe presence of the antigen and to determine the distribution of theantigen in the examined tissue. In situ detection can be accomplished byapplying a detectably-labeled structure to frozen or paraffin-embeddedtissue sections. General techniques of in situ detection are well-knownto those of ordinary skill. See, for example, Ponder, “Cell MarkingTechniques and Their Application,” in MAMMALIAN DEVELOPMENT: A PRACTICALAPPROACH 113-38 Monk (ed.) (IRL Press 1987), and Coligan at pages5.8.1-5.8.8.

Stably tethered structures can be detectably labeled with anyappropriate marker moiety, for example, a radioisotope, an enzyme, afluorescent label, a dye, a chromogen, a chemiluminescent label, abioluminescent label or a paramagnetic label.

The marker moiety may be a radioisotope that is detected by such meansas the use of a gamma counter or a beta-scintillation counter or byautoradiography. In a preferred embodiment, the diagnostic conjugate isa gamma-, beta- or a positron-emitting isotope. A marker moiety refersto a molecule that will generate a signal under predeterminedconditions. Examples of marker moieties include radioisotopes, enzymes,fluorescent labels, chemiluminescent labels, bioluminescent labels andparamagnetic labels. The binding of marker moieties to stably tetheredstructures can be accomplished using standard techniques known to theart. Typical methodology in this regard is described by Kennedy et al.,Clin. Chim. Acta 70: 1 (1976), Schurs et al., Clin. Chim. Acta 81: 1(1977), Shih et al., Intel J. Cancer 46: 1101 (1990).

Nucleic Acid Based In Vitro Diagnosis

Stably tethered structures may, in some embodiments, incorporatednucleic acid moieties. In particular embodiments, nucleic acids may beanalyzed to determine levels of binding, particularly using nucleic acidamplification methods. Various forms of amplification are well known inthe art and any such known method may be used. Generally, amplificationinvolves the use of one or more primers that hybridize selectively orspecifically to a target nucleic acid sequence to be amplified.

The term primer, as defined herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Computerized programs for selection anddesign of amplification primers are available from commercial and/orpublic sources well known to the skilled artisan. A number of templatedependent processes are available to amplify the marker sequencespresent in a given sample. One of the best-known amplification methodsis the polymerase chain reaction (referred to as PCR), which isdescribed in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159. However, other methods of amplification are known and may beused.

In Vivo Diagnosis

Methods of diagnostic imaging with labeled peptides or MAbs arewell-known. For example, in the technique of immunoscintigraphy, ligandsor antibodies are labeled with a gamma-emitting radioisotope andintroduced into a patient. A gamma camera is used to detect the locationand distribution of gamma-emitting radioisotopes. See, for example,Srivastava (ed.), RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING ANDTHERAPY (Plenum Press 1988), Chase, “Medical Applications ofRadioisotopes,” in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition,Gennaro et al. (eds.), pp. 624-652 (Mack Publishing Co., 1990), andBrown, “Clinical Use of Monoclonal Antibodies,” in BIOTECHNOLOGY ANDPHARMACY 227-49, Pezzuto et al. (eds.) (Chapman & Hall 1993). Alsopreferred is the use of positron-emitting radionuclides (PET isotopes),such as with an energy of 511 keV, such as ¹⁸F, ⁶⁸Ga, ⁶⁴Cu, and ¹²⁴I.Such imaging can be conducted by direct labeling of the stably tetheredstructure, or by a pretargeted imaging method, as described inGoldenberg et al, “Antibody Pre-targeting Advances CancerRadioimmunodetection and Radioimmunotherapy,” (J Clin Oncol 2006;24:823-834), see also U.S. Patent Publication Nos. 20050002945,20040018557, 20030148409 and 20050014207, each incorporated herein byreference.

The radiation dose delivered to the patient is maintained at as low alevel as possible through the choice of isotope for the best combinationof minimum half-life, minimum retention in the body, and minimumquantity of isotope which will permit detection and accuratemeasurement. Examples of radioisotopes that are appropriate fordiagnostic imaging include ^(99m)Tc and ¹¹¹In.

The stably tethered structures, or haptens or carriers that bind tothem, also can be labeled with paramagnetic ions and a variety ofradiological contrast agents for purposes of in vivo diagnosis. Contrastagents that are particularly useful for magnetic resonance imagingcomprise gadolinium, manganese, dysprosium, lanthanum, or iron ions.Additional agents include chromium, copper, cobalt, nickel, rhenium,europium, terbium, holmium, or neodymium. ligands, antibodies andfragments thereof can also be conjugated to ultrasoundcontrast/enhancing agents. For example, one ultrasound contrast agent isa liposome that comprises a humanized IgG or fragment thereof. Alsopreferred, the ultrasound contrast agent is a liposome that is gasfilled.

Imaging Agents and Radioisotopes

Many appropriate imaging agents are known in the art, as are methods fortheir attachment to proteins or peptides (see, e.g., U.S. Pat. Nos.5,021,236 and 4,472,509, both incorporated herein by reference). Certainattachment methods involve the use of a metal chelate complex employing,for example, an organic chelating agent such a DTPA attached to theprotein or peptide (U.S. Pat. No. 4,472,509). Proteins or peptides alsomay be reacted with an enzyme in the presence of a coupling agent suchas glutaraldehyde or periodate. Conjugates with fluorescein markers areprepared in the presence of these coupling agents or by reaction with anisothiocyanate.

Non-limiting examples of paramagnetic ions of potential use as imagingagents include chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and erbium (III), with gadolinium beingparticularly preferred. Ions useful in other contexts, such as X-rayimaging, include but are not limited to lanthanum (III), gold (III),lead (II), and especially bismuth (III).

Radioisotopes of potential use as imaging or therapeutic agents includeastatine²¹¹, carbon¹⁴, chromium⁵¹, chlorine³⁶, cobalt⁵⁷, cobalt⁵⁸,copper⁶², copper⁶⁴, copper⁶⁷, Eu¹⁵², fluorine¹⁸, gallium⁶⁷, gallium⁶⁸,hydrogen³, iodine¹²³, iodine¹²⁴, iodine¹²⁵, iodine¹³¹, indium¹¹¹,iron⁵², iron⁵⁹, lutetium¹⁷⁷, phosphorus³², phosphorus³³, rhenium¹⁸⁶,rhenium¹⁸⁸, Sc⁴⁷, selenium⁷⁵, silver¹¹¹, sulphur³⁵, technetium^(94m),technetium^(99m), yttrium⁸⁶ and yttrium⁹⁰, and zirconium⁸⁹. I¹²⁵ isoften being preferred for use in certain embodiments, andtechnetium^(99m) and indium¹¹¹ are also often preferred due to their lowenergy and suitability for long-range detection.

Radioactively labeled proteins or peptides may be produced according towell-known methods in the art. For instance, they can be iodinated bycontact with sodium or potassium iodide and a chemical oxidizing agentsuch as sodium hypochlorite, or an enzymatic oxidizing agent, such aslactoperoxidase. Proteins or peptides may be labeled withtechnetium-^(99m) by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the peptide to this column or bydirect labeling techniques, e.g., by incubating pertechnate, a reducingagent such as SNCl₂, a buffer solution such as sodium-potassiumphthalate solution, and the peptide. Intermediary functional groupswhich are often used to bind radioisotopes which exist as metallic ionsto peptides include diethylenetriaminepentaacetic acid (DTPA), DOTA,NOTA, porphyrin chelators and ethylene diaminetetracetic acid (EDTA).Also contemplated for use are fluorescent labels, including rhodamine,fluorescein isothiocyanate and renographin.

In certain embodiments, the proteins or peptides may be linked to asecondary binding ligand or to an enzyme (an enzyme tag) that willgenerate a colored product upon contact with a chromogenic substrate.Examples of suitable enzymes include urease, alkaline phosphatase,(horseradish) hydrogen peroxidase and glucose oxidase. Preferredsecondary binding ligands are biotin and avidin or streptavidincompounds. The use of such labels is well known to those of skill in theart in light and is described, for example, in U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241;each incorporated herein by reference. These fluorescent labels arepreferred for in vitro uses, but may also be of utility in in vivoapplications, particularly endoscopic or intravascular detectionprocedures.

In alternative embodiments, ligands, antibodies, or other proteins orpeptides may be tagged with a fluorescent marker. Non-limiting examplesof photodetectable labels include Alexa 350, Alexa 430, AMCA,aminoacridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G,BODIPY-TMR, BODIPY-TRX, 5-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein,5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxyrhodamine,6-carboxytetramethyl amino, Cascade Blue, Cy2, Cy3, Cy5,6-FAM, dansylchloride, Fluorescein, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1,3-diazole),Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue,phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet,cresyl blue violet, brilliant cresyl blue, para-aminobenzoic acid,erythrosine, phthalocyanines, azomethines, cyanines, xanthines,succinylfluoresceins, rare earth metal cryptates, europiumtrisbipyridine diamine, a europium cryptate or chelate, diamine,dicyanins, La Jolla blue dye, allopycocyanin, allococyanin B,phycocyanin C, phycocyanin R, thiamine, phycoerythrocyanin,phycoerythrin R, REG, Rhodamine Green, rhodamine isothiocyanate,Rhodamine Red, ROX, TAMRA, TET, TRIT (tetramethyl rhodamine isothiol),Tetramethylrhodamine, Edans and Texas Red. These and other luminescentlabels may be obtained from commercial sources such as Molecular Probes(Eugene, Oreg.), and EMD Biosciences (San Diego, Calif.).

Chemiluminescent labeling compounds of use may include luminol,isoluminol, an aromatic acridinium ester, an imidazole, an acridiniumsalt and an oxalate ester, or a bioluminescent compound such asluciferin, luciferase and aequorin. Diagnostic conjugates may be used,for example, in intraoperative, endoscopic, or intravascular tumor ordisease diagnosis.

In various embodiments, labels of use may comprise metal nanoparticles.Methods of preparing nanoparticles are known. (See e.g., U.S. Pat. Nos.6,054,495; 6,127,120; 6,149,868; Lee and Meisel, J. Phys. Chem.86:3391-3395, 1982.) Nanoparticles may also be obtained from commercialsources (e.g., Nanoprobes Inc., Yaphank, N.Y.; Polysciences, Inc.,Warrington, Pa.). Modified nanoparticles are available commercially,such as Nanogold® nanoparticles from Nanoprobes, Inc. (Yaphank, N.Y.).Functionalized nanoparticles of use for conjugation to proteins orpeptides may be commercially obtained.

EXAMPLES

The following examples are provided to illustrate, but not to limit theclaimed invention.

Methods for Generating Non-Covalent a₂b Complexes Composed of ThreeFab-Subunits Example 1 General Strategy for Production of Modular FabSubunits

Fab modules may be produced as fusion proteins containing either a DDDor AD sequence. Independent transgenic cell lines are developed for eachFab fusion protein. Once produced, the modules can be purified ifdesired or maintained in the cell culture supernatant fluid. Followingproduction, any (Fab-DDD)₂ module can be combined with any Fab-AD moduleto generate a bispecific trivalent Fab (bsTF).

The plasmid vector pdHL2 has been used to produce a number of antibodiesand antibody-based constructs. See Gillies et al., J Immunol Methods(1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6.The di-cistronic mammalian expression vector directs the synthesis ofthe heavy and light chains of IgG. The vector sequences are mostlyidentical for many different IgG-pdHL2 constructs, with the onlydifferences existing in the variable domain (VH and VL) sequences. Usingmolecular biology tools known to those skilled in the art, these IgGexpression vectors can be converted into Fab-DDD or Fab-AD expressionvectors. To generate Fab-DDD expression vectors, the coding sequencesfor the hinge, CH2 and CH3 domains of the heavy chain are replaced witha sequence encoding the first 4 residues of the hinge, a 14 residueGly-Ser linker and the first 44 residues of human RIIα (referred to asDDD1, FIG. 1). To generate Fab-AD expression vectors, the sequences forthe hinge, CH2 and CH3 domains of IgG are replaced with a sequenceencoding the first 4 residues of the hinge, a 15 residue Gly-Ser linkerand a 17 residue synthetic AD called AKAP-IS (referred to as AD1, FIG.2), which was generated using bioinformatics and peptide arraytechnology and shown to bind RIIα dimers with a very high affinity (0.4nM). See Alto, et al. Proc. Natl. Acad. Sci., U.S.A (2003), 100:4445-50.

Two shuttle vectors were designed to facilitate the conversion ofIgG-pdHL2 vectors to either Fab-DDD1 or Fab-AD1 expression vectors, asdescribed below.

Preparation of CH1

The CH1 domain was amplified by PCR using the pdHL2 plasmid vector as atemplate. The left PCR primer consists of the upstream (5′) of the CH1domain and a SacII restriction endonuclease site, which is 5′ of the CH1coding sequence. The right primer consists of the sequence coding forthe first 4 residues of the hinge (PKSC) followed by GGGGS with thefinal two codons (GS) comprising a Bam HI restriction site.

5′ of CH1 Left Primer (SEQ ID NO: 5) 5′GAACCTCGCGGACAGTTAAG-3′ CH1 +G₄S-Bam Right (SEQ ID NO: 6)5′GGATCCTCCGCCGCCGCAGCTCTTAGGTTTCTTGTCCACCTTGGTGTT GCTGG-3′

The 410 bp PCR amplimer was cloned into the pGemT PCR cloning vector(Promega, Inc.) and clones were screened for inserts in the T7 (5′)orientation.

Construction of (G₄S)₂DDD1

A duplex oligonucleotide, designated (G₄S)₂DDD1, was synthesized bySigma Genosys (Haverhill, UK) to code for the amino acid sequence ofDDD1 preceded by 11 residues of the linker peptide, with the first twocodons comprising a BamHI restriction site. A stop codon and an EagIrestriction site are appended to the 3′ end. The encoded polypeptidesequence is shown below.

(SEQ ID NO: 7) GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTR LREARA

The two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom,that overlap by 30 base pairs on their 3′ ends, were synthesized (SigmaGenosys) and combined to comprise the central 154 base pairs of the 174bp DDD1 sequence. The oligonucleotides were annealed and subjected to aprimer extension reaction with Taq polymerase.

RIIA1-44 top (SEQ ID NO: 8)5′GTGGCGGGTCTGGCGGAGGTGGCAGCCACATCCAGATCCCGCCGGGGCTCACGGAGCTGCTGCAGGGCTACACGGTGGAGGTGCTGCGACAG-3′ RIIA1-44 bottom(SEQ ID NO: 9) 5′GCGCGAGCTTCTCTCAGGCGGGTGAAGTACTCCACTGCGAATTCGACGAGGTCAGGCGGCTGCTGTCGCAGCACCTCCACCGTGTAGCCCTG-3′

Following primer extension, the duplex was amplified by PCR using thefollowing primers:

G4S Bam-Left (SEQ ID NO: 10) 5′-GGATCCGGAGGTGGCGGGTCTGGCGGAGGT-3′1-44 stop Eag Right (SEQ ID NO: 11) 5′-CGGCCGTCAAGCGCGAGCTTCTCTCAGGCG-3′

This amplimer was cloned into pGemT and screened for inserts in the T7(5′) orientation.

Construction of (G₄S)₂-AD1

A duplex oligonucleotide, designated (G₄S)₂-AD1, was synthesized (SigmaGenosys) to code for the amino acid sequence of AD1 preceded by 11residues of the linker peptide with the first two codons comprising aBamHI restriction site. A stop codon and an EagI restriction site areappended to the 3′ end. The encoded polypeptide sequence is shown below.

GSGGGGSGGGGSQIEYLAKQIVDNAIQQA (SEQ ID NO: 12)

Two complimentary overlapping oligonucleotides, designated AKAP-IS Topand AKAP-IS Bottom, were synthesized.

AKAP-IS Top (SEQ ID NO: 13)5′GGATCCGGAGGTGGCGGGTCTGGCGGAGGTGGCAGCCAGATCGAGTACCTGGCCAAGCAGATCGTGGACAACGCCATCCAGCAGGCCTGA CGGCCG-3′ AKAP-IS Bottom(SEQ ID NO: 14) 5′CGGCCGTCAGGCCTGCTGGATGGCGTTGTCCACGATCTGCTTGGCCAGGTACTCGATCTGGCTGCCACCTCCGCCAGACCCGCCACCTCC GGATCC-3′

The duplex was amplified by PCR using the following primers:

G4S Bam-Left (SEQ ID NO: 15) 5′-GGATCCGGAGGTGGCGGGTCTGGCGGAGGT-3′AKAP-IS stop Eag Right (SEQ ID NO: 16) 5′-CGGCCGTCAGGCCTGCTGGATG-3′

This amplimer was cloned into the pGemT vector and screened for insertsin the T7 (5′) orientation.

Ligating DDD1 with CH1

A 190 bp fragment encoding the DDD1 sequence was excised from pGemT withBamHI and NotI restriction enzymes and then ligated into the same sitesin CH1-pGemT to generate the shuttle vector CH1-DDD1-pGemT.

Ligating AD1 with CH1

A 110 bp fragment containing the AD1 sequence was excised from pGemTwith BamHI and NotI and then ligated into the same sites in CH1-pGemT togenerate the shuttle vector CH1-AD1-pGemT.

Cloning CH1-DDD1 or CH1-AD1 into pdHL2-Based Vectors

With this modular design either CH1-DDD1 or CH1-AD1 can be incorporatedinto any IgG construct in the pdHL2 vector. The entire heavy chainconstant domain is replaced with one of the above constructs by removingthe SacII/EagI restriction fragment (CH1-CH3) from pdHL2 and replacingit with the SacII/EagI fragment of CH1-DDD1 or CH1-AD1, which is excisedfrom the respective pGemT shuttle vector.

N-Terminal DDD Domains

The location of the DDD or AD is not restricted to the carboxyl terminalend of CH1. A construct was engineered in which the DDD1 sequence wasattached to the amino terminal end of the VH domain.

Example 2 Expression Vectors

Construction of h679-Fd-AD1-pdHL2

h679-Fd-AD1-pdHL2 is an expression vector for production of h679 Fabwith AD1 coupled to the carboxyl terminal end of the CH1 domain of theFd via a flexible Gly/Ser peptide spacer composed of 14 amino acidresidues. A pdHL2-based vector containing the variable domains of h679was converted to h679-Fd-AD1-pdHL2 by replacement of the SacII/EagIfragment with the CH1-AD1 fragment, which was excised from theCH1-AD1-SV3 shuttle vector with SacII and EagI.

Construction of C-DDD1-Fd-hMN-14-pdHL2

C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of astable dimer that comprises two copies of a fusion proteinC-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the carboxylterminus of CH1 via a flexible peptide spacer. The plasmid vectorhMN14(I)-pdHL2, which has been used to produce hMN-14 IgG, was convertedto C-DDD1-Fd-hMN-14-pdHL2 by digestion with SacII and EagI restrictionendonucleases to remove the CH1-CH3 domains and insertion of theCH1-DDD1 fragment, which was excised from the CH1-DDD1-SV3 shuttlevector with SacII and EagI.

Construction of N-DDD1-Fd-hMN-14-pdHL2

N-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of astable dimer that comprises two copies of a fusion proteinN-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the aminoterminus of VH via a flexible peptide spacer.

The expression vector was engineered as follows. The DDD1 domain wasamplified by PCR using the two primers shown below.

DDD1 Nco Left (SEQ ID NO: 17) 5′CCATGGGCAGCCACATCCAGATCCCGCC-3′DDD1-G₄S Bam Right (SEQ ID NO: 18)5′GGATCCGCCACCTCCAGATCCTCCGCCGCCAGCGCGAGCTTCTCTC AGGCGGGTG-3′

As a result of the PCR, an NcoI restriction site and the coding sequencefor part of the linker (G₄S)₂ containing a BamHI restriction wereappended to the 5′ and 3′ ends, respectively. The 170 bp PCR amplimerwas cloned into the pGemT vector and clones were screened for inserts inthe T7 (5′) orientation. The 194 bp insert was excised from the pGemTvector with NcoI and SalI restriction enzymes and cloned into the SV3shuttle vector, which was prepared by digestion with those same enzymes,to generate the intermediate vector DDD1-SV3.

The hMN-14 Fd sequence was amplified by PCR using the oligonucleotideprimers shown below.

hMN-14VH left G4S Bam (SEQ ID NO: 19)5′-GGATCCGGCGGAGGTGGCTCTGAGGTCCAACTGGTGGAGAGCGG-3′ CH1-C stop Eag(SEQ ID NO: 20) 5′-CGGCCGTCAGCAGCTCTTAGGTTTCTTGTC-3′

As a result of the PCR, a BamHI restriction site and the coding sequencefor part of the linker (G₄S) were appended to the 5′ end of theamplimer. A stop codon and EagI restriction site was appended to the 3′end. The 1043 bp amplimer was cloned into pGemT. The hMN-14-Fd insertwas excised from pGemT with BamHI and EagI restriction enzymes and thenligated with DDD1-SV3 vector, which was prepared by digestion with thosesame enzymes, to generate the construct N-DDD1-hMN-14Fd-SV3.

The N-DDD1-hMN-14 Fd sequence was excised with XhoI and EagI restrictionenzymes and the 1.28 kb insert fragment was ligated with a vectorfragment that was prepared by digestion of C-hMN-14-pdHL2 with thosesame enzymes. The final expression vector is N-DDD1-Fd-hMN-14-pDHL2.

Example 3 Production and Purification of h679-Fab-AD1

The h679-Fd-AD1-pdHL2 vector was linearized by digestion with Sal Irestriction endonuclease and transfected into Sp/EEE myeloma cells byelectroporation. The di-cistronic expression vector directs thesynthesis and secretion of both h679 kappa light chain and h679 Fd-AD1,which combine to form h679 Fab-AD1. Following electroporation, the cellswere plated in 96-well tissue culture plates and transfectant cloneswere selected with 0.05 μM methotrexate (MTX). Clones were screened forprotein expression by ELISA using microtitre plates coated with aBSA-IMP-260 (HSG) conjugate and detection with HRP-conjugated goatanti-human Fab. BIAcore analysis using an HSG (IMP-239) sensorchip wasused to determine the productivity by measuring the initial slopeobtained from injection of diluted media samples. The highest producingclone had an initial productivity of approximately 30 mg/L. A total of230 mg of h679-Fab-AD1 was purified from 4.5 liters of roller bottleculture by single-step IMP-291 affinity chromatography. Culture mediawas concentrated approximately 10-fold by ultrafiltration before loadingonto an IMP-291-affigel column. The column was washed to baseline withPBS and h679-Fab-AD1 was eluted with 1 M imidazole, 1 mM EDTA, 0.1 MNaAc, pH 4.5. SE-HPLC analysis of the eluate showed a single sharp peakwith a retention time (9.63 min) consistent with a 50 kDa protein (notshown). Only two bands, which represent the polypeptide constituents ofh679-AD1, were evident by reducing SDS-PAGE analysis (not shown).

Example 4 Production and Purification of N-DDD1-Fab-hMN-14 andC-DDD1-Fab-hMN-14

The C-DDD1-Fd-hMN-14-pdHL2 and N-DDD1-Fd-hMN-14-pdHL2 vectors weretransfected into Sp2/0-derived myeloma cells by electroporation.C-DDD1-Fd-hMN-14-pdHL2 is a di-cistronic expression vector, whichdirects the synthesis and secretion of both hMN-14 kappa light chain andhMN-14 Fd-DDD1, which combine to form C-DDD1-hMN-14 Fab.N-DDD1-hMN-14-pdHL2 is a di-cistronic expression vector, which directsthe synthesis and secretion of both hMN-14 kappa light chain andN-DDD1-Fd-hMN-14, which combine to form N-DDD1-Fab-hMN-14. Each fusionprotein forms a stable homodimer via the interaction of the DDD1 domain.

Following electroporation, the cells were plated in 96-well tissueculture plates and transfectant clones were selected with 0.05 μMmethotrexate (MTX). Clones were screened for protein expression by ELISAusing microtitre plates coated with WI2 (a rat anti-id monoclonalantibody to hMN-14) and detection with HRP-conjugated goat anti-humanFab. The initial productivity of the highest producing C-DDD1-Fab-hMN14Fab and N-DDD1-Fab-hMN14 Fab clones was 60 mg/L and 6 mg/L,respectively.

Affinity Purification of N-DDD1-hMN-14 and C-DDD1-hMN-14 withAD1-Affigel

The DDD/AD interaction was utilized to affinity purify DDD1-containingconstructs. AD1-C is a peptide that was made synthetically consisting ofthe AD1 sequence and a carboxyl terminal cysteine residue (see Example6), which was used to couple the peptide to Affigel following reactionof the sulfhydryl group with chloroacetic anhydride. DDD-containing a₂structures specifically bind to the AD1-C-Affigel resin at neutral pHand can be eluted at low pH (e.g., pH 2.5).

A total of 81 mg of C-DDD1-Fab-hMN-14 was purified from 1.2 liters ofroller bottle culture by single-step AD1-C affinity chromatography.Culture media was concentrated approximately 10-fold by ultrafiltrationbefore loading onto an AD1-C-affigel column. The column was washed tobaseline with PBS and C-DDD1-Fab-hMN-14 was eluted with 0.1 M Glycine,pH 2.5. SE-HPLC analysis of the eluate showed a single protein peak witha retention time (8.7 min) consistent with a 107 kDa protein (notshown). The purity was also confirmed by reducing SDS-PAGE, showing onlytwo bands of molecular size expected for the two polypeptideconstituents of C-DDD1-Fab-hMN-14 (not shown).

A total of 10 mg of N-DDD1-hMN-14 was purified from 1.2 liters of rollerbottle culture by single-step AD1-C affinity chromatography as describedabove. SE-HPLC analysis of the eluate showed a single protein peak witha retention time (8.77 min) similar to C-DDD1-Fab-hMN-14 and consistentwith a 107 kDa protein (not shown). Reducing SDS-PAGE showed only twobands attributed to the polypeptide constituents of N-DDD1-Fab-hMN-14(not shown).

The binding activity of C-DDD1-Fab-hMN-14 was determined by SE-HPLCanalysis of samples in which the test article was mixed with variousamounts of WI2. A sample prepared by mixing WI2 Fab andC-DDD1-Fab-hMN-14 at a molar ratio of 0.75:1 showed three peaks, whichwere attributed to unbound C-DDD1-Fab-hMN14 (8.71 min),C-DDD1-Fab-hMN-14 bound to one WI2 Fab (7.95 min), and C-DDD1-Fab-hMN14bound to two WI2 Fabs (7.37 min) (not shown). When a sample containingWI2 Fab and C-DDD1-Fab-hMN-14 at a molar ratio of 4 was analyzed, only asingle peak at 7.36 minutes was observed (not shown). These resultsdemonstrate that hMN14-Fab-DDD1 is dimeric and has two active bindingsites. Very similar results were obtained when this experiment wasrepeated with N-DDD1-Fab-hMN-14.

A competitive ELISA demonstrated that both C-DDD1-Fab-hMN-14 andN-DDD1-Fab-hMN-14 bind to CEA with an avidity similar to hMN-14 IgG, andsignificantly stronger than monovalent hMN-14 Fab (not shown). ELISAplates were coated with a fusion protein containing the epitope (A3B3)of CEA for which hMN-14 is specific.

Example 5 Formation of a₂b Complexes

Evidence for the formation of an a₂b complex was first provided bySE-HPLC analysis of a mixture containing C-DDD1-Fab-hMN-14 (as a₂) andh679-Fab-AD1 (as b) in an equal molar amount. When such a sample wasanalyzed, a single peak was observed having a retention time of 8.40minutes, which is consistent with the formation of a new protein that islarger than either h679-Fab-AD1 (9.55 min) or C-DDD1-Fab-hMN-14 (8.73min) alone (not shown). The upfield shift was not observed when hMN-14F(ab′)₂ was mixed with h679-Fab-AD1 or C-DDD1-Fab-hMN-14 was mixed with679-Fab-NEM, demonstrating that the interaction is mediated specificallyvia the DDD1 and AD1 domains. Very similar results were obtained usingh679-Fab-AD1 and N-DDD1-Fab-hMN-14 (not shown).

BIAcore was used to further demonstrate and characterize the specificinteraction between the DD1 and AD1 fusion proteins. The experimentswere performed by first allowing either h679-Fab-AD1 or 679-Fab-NEM tobind to the surface of a high density HSG-coupled (IMP239) sensorchip,followed by a subsequent injection of C-DDD1-Fab-hMN-14 or hMN-14F(ab′)₂. As expected, only the combination of h679-Fab-AD1 andC-DDD1-Fab-hMN-14 resulted in a further increase in response units whenthe latter was injected (not shown). Similar results were obtained usingN-DDD1-Fab-hMN-14 and h679-Fab-AD1 (not shown).

Equilibrium SE-HPLC experiments were carried out to determine thebinding affinity of the specific interaction between AD1 and DDD1present in the respective fusion proteins. The dissociation constants(K_(d)) for the binding of h679-Fab-AD1 with C-DDD1-Fab-hMN-14,N-DDD1-hMN-14 and a commercial sample of recombinant human RIIα werefound to be 15 nM, 8 nM and 30 nM, respectively.

Other Related Methods Example 6 Generation of Di-AD1

In this example, a small polypeptide (AD1-C) having the amino acidsequence shown below was made synthetically.

NH₂-KQIEYLAKQIVDNAIQQAKGC-COOH (SEQ ID NO: 37)

In AD1-C, the AD1 amino acid sequence (underlined) is flanked by alysine residue at N-terminus and a KGC tripeptide at carboxyl terminus.Two lysine (K) residues were introduced to increase solubility and aglycine (G) residue was inserted before the C-terminal cysteine toprovide added flexibility. Upon treatment with DMSO, AD1-C was oxidizedto a dimer, designated Di-AD1, which was purified by RP-HPLC. Aschematic structure of Di-AD1 is shown below (=indicating the disulfidebridge)

(SEQ ID NO: 21) NH₂-KQIEYLAKQIVDNAIQQAKGC = CGKAQQIANDVIQKALYEIQ K-NH₂

There are a number of functional groups present in Di-AD1 or AD1-C thatmay be utilized for further modifications. For examples, the 8 and 4primary amino groups contained in Di-AD1 and AD1-C, respectively, may beused to couple drugs, toxins, proteins, or other effectors. Further,Di-AD1 and AD1-C have 2 and 1 tyrosine residues, respectively, which maybe used for radio-iodination. Finally, AD1-C contains a free cysteineresidue, which can also be used to couple effectors or form a Di-AD1analog containing effectors.

Example 7 A Novel Pretargeting Approach

The method of the present invention lends itself to new pretargetingmethodologies.

The following provides an example of a pretargeting system that uses theaffinity enhancement system (Le Doussal et al., J Nucl Med (1989),30:1358-66) without the need for a hapten-binding antibody. A dimer ofC-DDD1-Fab-hMN-14 or N-Fab-DDD2-hMN-14, produced as described in Example4, may be used for pretargeting a tumor. The 107 kDa protein is firstadministered intravenously to patients and allowed to bind CEA on tumorswhile clearing from blood and normal tissues. At a later time, adivalent peptide, such as a DOTA conjugate of Di-AD1 carrying atherapeutic (for example, ⁹⁰Y) or diagnostic radioisotope (for example,¹¹¹In), is administered intravenously. The small peptide (=5000 Da),while rapidly clearing from blood and normal tissues, localizes to thetumor as it contains two AD sequences that is expected to interactspecifically with C-DDD1-Fab-hMN-14 already retained by the tumor.

Cross-linking of C-DDD1-Fab-hMN-14 with Di-AD1 in vitro was demonstratedby SE-HPLC. When C-DDD1-Fab-hMN-14 was mixed with Di-AD1 the proteinpeak shifted from 8.67 min to 7.95 min, indicating the formation of acrosslinked structure (not shown). No such shift was observed whenhMN-14 F(ab′)₂ was mixed with Di-AD1, demonstrating that thecross-linking is mediated by the interaction between DDD1 and AD1. Toconfirm that the peak shift was in fact due to specific cross-linking ofC-DDD1-Fab-hMN-14, the complex was reduced with DTT to cleave thedisulfide linkage of Di-AD1, which resulted in the shift of the peakback to 8.67 min (not shown).

Example 8 Affinity Purification of Either DDD or AD Fusion Proteins

Universal affinity purification systems can be developed by productionof DDD or AD proteins, which have lower affinity docking. The DDD formedby RIα dimers binds AKAP-IS (AD1) with a 500-fold weaker affinity (225nM) compared to RIIα. Thus, RIα dimers formed from the first 44 aminoacid resides can be produced and coupled to a resin to make anattractive affinity matrix for purification of any AD1-containing fusionprotein.

Many lower affinity (0.1 μM) AKAP anchoring domains exist in nature. Ifnecessary, highly predicable amino acid substitutions can be introducedto further lower the binding affinity. A low affinity AD can be producedeither synthetically or biologically and coupled to resin for use inaffinity purification of any DDD1 fusion protein.

Methods Related to the Generation of Stably Tethered Structures Example9 Vectors for Producing Disulfide Stabilized Structures Composed ofThree Fab Fragments

N-DDD2-Fd-hMN-14-pdHL2

N-DDD2-hMN-14-pdHL2 is an expression vector for production ofN-DDD2-Fab-hMN-14, which possesses a dimerization and docking domainsequence of DDD2 appended to the amino terminus of the Fd (FIG. 4). TheDDD2 is coupled to the V_(H) domain via a 15 amino acid residue Gly/Serpeptide linker. DDD2 has a cysteine residue preceding the dimerizationand docking sequences, which are identical to those of DDD1. The fusionprotein secreted is composed of two identical copies of hMN-14 Fab heldtogether by non-covalent interaction of the DDD2 domains (FIG. 3B).

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides (DDD2 Top and DDD2 Bottom), whichcomprise residues 1-13 of DDD2, were made synthetically. Theoligonucleotides were annealed and phosphorylated with T4 polynucleotidekinase (PNK), resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases NcoI and PstI, respectively.

DDD2 Top (SEQ ID NO: 22) 5′CATGTGCGGCCACATCCAGATCCCGCCGGGGCTCACGGAGCTGCTGCA-3′ DDD2 Bottom (SEQ ID NO: 23)5′GCAGCTCCGTGAGCCCCGGCGGGATCTGGATGTGGCCGCA-3′

The duplex DNA was ligated with a vector fragment, DDD1-hMN14 Fd-SV3that was prepared by digestion with NcoI and PstI, to generate theintermediate construct DDD2-hMN14 Fd-SV3. A 1.28 kb insert fragment,which contained the coding sequence for DDD2-hMN14 Fd, was excised fromthe intermediate construct with XhoI and EagI restriction endonucleasesand ligated with hMN14-pdHL2 vector DNA that was prepared by digestionwith those same enzymes. The final expression vector isN-DDD2-Fd-hMN-14-pdHL2 (FIG. 4).

C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production ofC-DDD2-Fab-hMN-14, which possesses a dimerization and docking domainsequence of DDD2 appended to the carboxyl terminus of the Fd via a 14amino acid residue Gly/Ser peptide linker (FIG. 5A). The fusion proteinsecreted is composed of two identical copies of hMN-14 Fab held togetherby non-covalent interaction of the DDD2 domains (FIG. 5B).

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides, which comprise the coding sequence forpart of the linker peptide (GGGGSGGGCG, SEQ ID NO:38) and residues 1-13of DDD2, were made synthetically. The oligonucleotides were annealed andphosphorylated with T4 PNK, resulting in overhangs on the 5′ and 3′ endsthat are compatible for ligation with DNA digested with the restrictionendonucleases BamHI and PstI, respectively.

G4S-DDD2 top (SEQ ID NO: 24)5′GATCCGGAGGTGGCGGGTCTGGCGGAGGTTGCGGCCACATCCAGATCCCGCCGGGGCTCACGGAGCTGCTGCA-3′ G4S-DDD2 bottom (SEQ ID NO: 25)5′GCAGCTCCGTGAGCCCCGGCGGGATCTGGATGTGGCCGCAACCTC CGCCAGACCCGCCACCTCCG-3′

The duplex DNA was ligated with the shuttle vector CH1-DDD1-pGemT, whichwas prepared by digestion with BamHI and PstI, to generate the shuttlevector CH1-DDD2-pGemT. A 507 bp fragment was excised from CH1-DDD2-pGemTwith SacII and EagI and ligated with the IgG expression vectorhMN14(I)-pdHL2, which was prepared by digestion with SacII and EagI. Thefinal expression construct is C-DDD2-Fd-hMN-14-pdHL2 (FIG. 6)

h679-Fd-AD2-pdHL2

h679-Fab-AD2, was designed to pair as B to N-DDD2-Fab-hMN-14 orC-DDD2-Fab-hMN-14. h679-Fd-AD2-pdHL2 is an expression vector for theproduction of h679-Fab-AD2, which possesses an anchor domain sequence ofAD2 appended to the carboxyl terminal end of the CH1 domain via a 14amino acid residue Gly/Ser peptide linker. AD2 has one cysteine residuepreceding and another one following the anchor domain sequence of AD1.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides (AD2 Top and AD2 Bottom), which comprisethe coding sequence for AD2 and part of the linker sequence, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and SpeI, respectively.

AD2 Top (SEQ ID NO: 26) 5′GATCCGGAGGTGGCGGGTCTGGCGGATGTGGCCAGATCGAGTACCTGGCCAAGCAGATCGTGGACAACGCCATCCAGCAGGCCGGCTGCTGA A-3′ AD2 Bottom(SEQ ID NO: 27) 5′TTCAGCAGCCGGCCTGCTGGATGGCGTTGTCCACGATCTGCTTGGCCAGGTACTCGATCTGGCCACATCCGCCAGACCCGCCACCTCCG-3′

The duplex DNA was ligated into the shuttle vector CH1-AD1-pGemT, whichwas prepared by digestion with BamHI and SpeI, to generate the shuttlevector CH1-AD2-pGemT. A 429 base pair fragment containing CH1 and AD2coding sequences was excised from the shuttle vector with SacII and EagIrestriction enzymes and ligated into h679-pdHL2 vector that prepared bydigestion with those same enzymes. The final expression vector ish679-Fd-AD2-pdHL2.

Example 10 Production of h679-Fab-AD2

The h679-Fd-AD2-pdHL2 vector was transfected into Sp/EEE myeloma cellsby electroporation. The di-cistronic expression vector directs thesynthesis and secretion of both h679 kappa light chain and h679 Fd-AD2,which combine to form h679-Fab-AD2. The cysteine residues on either endof the AD provide two potentially reactive sulfhydryl groups. Followingelectroporation, the cells were plated in 96-well tissue culture platesand transfectant clones were selected with 0.05 μM methotrexate (MTX).Clones were screened for protein expression by ELISA using microtitreplates coated with a BSA-IMP-260 (HSG) conjugate and detection with goatanti-human Fab-HRP. BIAcore analysis using an HSG (IMP-239) sensorchipwas used to determine the productivity by measuring the initial slopeobtained from injection of diluted media samples. The highest producingclone had an initial productivity of approximately 50 mg/L. A total of160 mg of h679-Fab-AD2 was purified from 2.9 liters of roller bottleculture by single-step IMP-291 affinity chromatography. Culture mediawas concentrated approximately 10-fold by ultrafiltration before loadingonto an IMP-291-affigel column. The column was washed to baseline withPBS and h679-AD2 was eluted with 1 M imidazole, 1 mM EDTA, 0.1 M NaAc,pH 4.5. SE-HPLC analysis shows a single sharp peak with a retention time(˜10 min) consistent with a 50 kDa protein (not shown). When thismaterial was mixed with hMN14-Fab-DDD1, only ⅓ was reactive as evidentby the observation in the SE-HPLC trace of a new peak attributed to thebinary complex (not shown). However, reduction of the h679-Fab-AD2 withTCEP resulted in 100% activity (not shown). This suggests that (1) anintramolecular disulfide bond may form between the two cysteine residuesof AD2, preventing association with the DDD but also protecting thesulfhydryl groups from reacting with other substances; and (2) theintramolecular disulfide bridge can be broken by reduction resulting ina DDD-reactive anchor domain with two free sufhydryl groups.

Example 11 Production of N-DDD2-Fab-hMN-14 as an a₂ Structure

The N-DDD2-Fd-hMN-14-pdHL2 vector was transfected into Sp/EEE myelomacells by electroporation. The di-cistronic expression vector directs thesynthesis and secretion of both hMN-14 kappa light chain andN-DDD2-hMN-14 Fd, which combine to form N-DDD2-hMN14 Fab. An A₂structure is expected to form by dimerization via DDD2, resulting in twopotentially reactive sulfhydryl groups provided by the cysteine residuein each DDD2. Following electroporation, the cells were plated in96-well tissue culture plates and transfectant clones were selected with0.05 μM methotrexate (MTX).

Clones were screened for protein expression by ELISA using microtitreplates coated with WI2 (hMN-14 anti-Id) and detection with goatanti-human Fab-HRP. The highest producing clones had an initialproductivity of approximately 10 mg/L. A total of 16 mg of N-DDD2-hMN-14was purified by protein L affinity chromatography from 1.8 liters ofroller bottle culture. Culture media was concentrated approximately10-fold by ultrafiltration before loading onto a protein L affinitychromatography column. The column was washed to baseline with PBS andN-DDD2-hMN14 was eluted with 1 mM EDTA, 0.1 M NaAc, pH 2.5 andimmediately neutralized with Tris-HCl. SE-HPLC analysis showed fourprotein peaks (not shown), two of which were subsequently identified asa₄ (7.9 min) and a₂ (8.8 min) forms of N-DDD2-Fab-hMN-14 and theremaining two were the dimer and monomer of the kappa chain. Thismixture showed little binding activity with h679-Fab-AD1, unless athiol-reducing agent such as TCEP was added to convert most of the a₄form to the a₂ form (not shown). These data suggest that (1) a₄ isformed via linking two a₂ structures through the cysteines present inthe DDD2, thereby preventing association with the AD but also protectingthe sulfhydryl groups from reacting with other substances; and (2) theintermolecular disulfide bridges can be broken by reduction, resultingin an a₂ structures with AD-reactive DDD dimers containing two freesufhydril groups. Note that this side-product (a₄) is composed of fouractive Fab subunits. Approximately 15% of the total N-DDD2-Fab-hMN-14remains in the A₄ form following reduction, even with high TCEPconcentrations and long reaction times (not shown). This suggests thatother mechanisms such as domain swapping may contribute to the formationof the a₄ form, in addition to disulfide bridging.

Example 12 Production of C-DDD2-Fab-hMN-14

The C-DDD2-Fd-hMN-14-pdHL2 vector was transfected into Sp/EEE myelomacells by electroporation. The di-cistronic expression vector directs thesynthesis and secretion of both hMN-14 kappa light chain andC-DDD2-Fd-hMN-14, which combine to form C-DDD2-Fab-hMN14. LikeN-DDD2-Fab-hMN-14, an a₂ structure is expected to form by dimerizationvia DDD2, resulting in two potentially reactive sulfhydryl groupsprovided by the cysteine residue in each DDD2. Followingelectroporation, the cells were plated in 96-well tissue culture platesand transfectant clones were selected with 0.05 μM methotrexate (MTX).

Clones were screened for protein expression by ELISA using microtitreplates coated with WI2 (hMN-14 anti-Id) and detection with goatanti-human Fab-HRP. The highest producing clones had an initialproductivity of approximately 100 mg/L, which was 10-fold higher thanthat of N-DDD2-Fab-hMN-14. A total of 200 mg of C-DDD2-hMN-14 waspurified by protein L affinity chromatography from 1.8 liters of rollerbottle culture as described in Example 3. The SE-HPLC profile of theProtein L-purified C-DDD2-Fab-hMN-14 (not shown) was similar to that ofN-DDD2-Fab-hMN-14. Two of the four protein peaks were identified as thea₄ (8.40 min) and a₂ (9.26 min) forms of C-DDD2-Fab-hMN-14 and theremaining two represent dimer and monomer of the kappa chain. Thismixture showed little binding activity with h679-AD1, unless athiol-reducing agent such as TCEP was added to convert most of the a₄form of to the a₂ form, which then bound avidly to h679-AD1. These datasuggest that C-DDD2-Fab-hMN-14 is a functional equivalent ofN-DDD2-hMN-14.

Example 13 Generation of TF1

h679-Fab-AD2 was designed as a B component to pair with an a₂ componentsuch as N-DDD2-hMN-Fab-14 or C-DDD2-hMN-14, which when combined, wouldreadily associate to form an a₂b structure (FIG. 7A) that might befurther induced to bind covalently via disulfide bonds (FIG. 7B). Sincecharacterization of N-DDD2- and AD2-constructs demonstrated thatreduction of each was necessary to achieve full DDD/AD interaction, areduction step was included in the process. Initially, immobilized TCEPwas used as a reducing agent to save the time required for removal ofthe reducing agent. Following reduction for 1 hour at room temperature,the TCEP-Agarose was removed by centrifugation and DMSO was added to thereaction solution to a final concentration of 10%. The first evidence ofthe existence of a covalently linked a₂b complex, henceforth referred toas TF1, was demonstrated by BIAcore analysis.

After feasibility was established in small-scale reactions usingimmobilized TCEP, a large scale preparation of TF1 was carried out asfollows. N-DDD2-Fab-hMN-14 (Protein L-purified) and h679-Fab-AD2(IMP-291-purified) were first mixed in roughly stoichiometricconcentrations in 1 mM EDTA, PBS, pH 7.4. Before the addition of TCEP,SE-HPLC did not show any evidence of a₂b formation (not shown). Insteadthere were peaks representing a₄ (7.97 min; 200 kDa), a₂ (8.91 min; 100kDa) and B (10.01 min; 50 kDa). Addition of 5 mM TCEP rapidly resultedin the formation of the a₂b complex as demonstrated by a new peak at8.43 min, consistent with a 150 kDa protein (not shown). Apparentlythere was excess B in this experiment as a peak attributed toh679-Fab-AD2 (9.72 min) was still evident yet no apparent peakcorresponding to either a₂ or a₄ was observed (not shown). Afterreduction for one hour, the TCEP was removed by overnight dialysisagainst several changes of PBS. The resulting solution was brought to10% DMSO and held overnight at room temperature.

When analyzed by SE-HPLC, the peak representing a₂b appeared to besharper with a slight reduction of the retention time by 0.1 min to 8.31min (not shown), which, based on our previous findings, indicates anincrease in binding affinity. The complex was further purified byIMP-291 affinity chromatography to remove the kappa chain contaminants.As expected, the excess h679-AD2 was co-purified and later removed bypreparative SE-HPLC (not shown).

TF1 is a highly stable complex. When TF1 was tested for binding to anHSG (IMP-239) sensorchip, there was no apparent decrease of the observedresponse at the end of sample injection. In contrast, when a solutioncontaining an equimolar mixture of both C-DDD1-Fab-hMN-14 andh679-Fab-AD1 was tested under similar conditions, the observed increasein response units was accompanied by a detectable drop during andimmediately after sample injection, indicating that the initially formeda₂b structure was unstable. Moreover, whereas subsequent injection ofWI2 gave a substantial increase in response units for TF1, no increasewas evident for the C-DDD1/AD1 mixture.

The additional increase of response units resulting from the binding ofWI2 to TF1 immobilized on the sensorchip corresponds to two fullyfunctional binding sites, each contributed by one subunit ofN-DDD2-Fab-hMN-14. This was confirmed by the ability of TF1 to bind twoFab fragments of WI2 (not shown). When a mixture containing h679-AD2 andN-DDD1-hMN14, which had been reduced and oxidized exactly as TF1, wasanalyzed by BIAcore, there was little additional binding of WI2 (notshown), indicating that a disulfide-stabilized a₂b complex such as TF1could only form through the interaction of DDD2 and AD2.

Two improvements to the process were implemented to reduce the time andefficiency of the process. First, a slight molar excess ofN-DDD2-Fab-hMN-14 present as a mixture of a₄/a₂ structures was used toreact with h679-Fab-AD2 so that no free h679-Fab-AD2 remained and anya₄/a₂ structures not tethered to h679-Fab-AD2, as well as light chains,would be removed by IMP-291 affinity chromatography. Second, hydrophobicinteraction chromatography (HIC) has replaced dialysis or diafiltrationas a means to remove TCEP following reduction, which would not onlyshorten the process time but also add a potential viral removing step.N-DDD2-Fab-hMN-14 and 679-Fab-AD2 were mixed and reduced with 5 mM TCEPfor 1 hour at room temperature. The solution was brought to 0.75 Mammonium sulfate and then loaded onto a Butyl FF HIC column. The columnwas washed with 0.75 M ammonium sulfate, 5 mM EDTA, PBS to remove TCEP.The reduced proteins were eluted from the HIC column with PBS andbrought to 10% DMSO. Following incubation at room temperature overnight,highly purified TF1 was isolated by IMP-291 affinity chromatography (notshown). No additional purification steps, such as gel filtration, wererequired.

Example 14 Generation of TF2

Following the successful creation of TF1, an analog designated TF2 (FIG.8) was also obtained by reacting C-DDD2-Fab-hMN-14 with h679-Fab-AD2.TF2 has two potential advantages over TF1. First, C-DDD2-Fab-hMN-14 isproduced at a 10-fold higher level than N-DDD2-Fab-hMN-14. Secondly,fusion proteins with C-terminal DDD domains exhibit a markedly strongerCEA-binding avidity than those with N-terminal DDD domains. This islikely attributed to the arrangement of the domains, where the bindingof the N-DDD variants may be compromised due to steric interference.

A pilot batch of TF2 was generated with >90% yield as follows. ProteinL-purified C-DDD2-Fab-hMN-14 (200 mg) was mixed with h679-Fab-AD2 (60mg) at a 1.4:1 molar ratio. The total protein concentration was 1.5mg/ml in PBS containing 1 mM EDTA. Subsequent steps involving TCEPreduction, HIC chromatography, DMSO oxidation, and IMP-291 affinitychromatography were the same as described for TF1. Before the additionof TCEP, SE-HPLC did not show any evidence of a₂b formation (not shown).Instead there were peaks corresponding to a₄ (8.40 min; 215 kDa), a₂(9.32 min; 107 kDa) and b (10.33 min; 50 kDa). Addition of 5 mM TCEPrapidly resulted in the formation of a₂b complex as demonstrated by anew peak at 8.77 min (not shown), consistent with a 157 kDa proteinexpected for the binary structure. TF2 was purified to near homogeneityby IMP-291 affinity chromatography (not shown). SE-HPLC analysis of theIMP-291 unbound fraction demonstrates the removal of a₄, a₂ and freekappa chains from the product (not shown).

Non-reducing SDS-PAGE analysis demonstrated that the majority of TF2exists as a large, covalent structure with a relative mobility near thatof IgG (not shown). The additional bands suggest that disulfideformation is incomplete under the experimental conditions. ReducingSDS-PAGE shows that any additional bands apparent in the non-reducinggel are product-related (not shown), as only bands representing theconstituent polypeptides of TF2 are evident. However, the relativemobilities of each of the four polypeptides are too close to beresolved. MALDI-TOF mass spectrometry (not shown) revealed a single peakof 156,434 Da, which is within 99.5% of the calculated mass (157,319 Da)of TF2.

The functionality of TF2 was determined by BIACORE as described for TF1.TF2, C-DDD1-hMN-14+h679-AD1 (used as a control sample of noncovalent a₂bcomplex), or C-DDD2-hMN-14+h679-AD2 (used as a control sample ofunreduced a₂ and b components) were diluted to 1 μg/ml (total protein)and pass over a sensorchip immoblized with HSG. The response for TF2 wasapproximately two-fold that of the two control samples, indicating thatonly the h679-Fab-AD component in the control samples would bind to andremains on the sensorchip. Subsequent WI2 IgG injections demonstratedthat only TF2 had a DDD-Fab-hMN-14 component that was tightly associatedwith h679-Fab-AD as indicated by an additional signal response. Theadditional increase of response units resulting from the binding of WI2to TF2 immobilized on the sensorchip also corresponds to two fullyfunctional binding sites, each contributed by one subunit ofC-DDD2-Fab-hMN-14. This was confirmed by the ability of TF2 to bind twoFab fragments of WI2 (not shown).

The relative CEA-binding avidity of TF2 was determined by competitiveELISA. Plates were coated (0.5 μg/well) with a fusion protein containingthe A3B3 domain of CEA, which is recognized by hMN-14. Serial dilutionsof TF1, TF2 and hMN-14 IgG were made in quadruplicate and incubated inwells containing HRP-conjugated hMN-14 IgG (1 nM). The data indicatethat TF2 binds CEA with an avidity that is at least equivalent to thatof IgG and two-fold stronger than TF1 (not shown). This is notsurprising since previously in a similar assay, C-DDD1-Fab-hMN-14 wasfound to have a stronger CEA-binding avidity than hMN-14 IgG, which inturn bound more avidly than N-DDD1-Fab-hMN-14. A possible explanationfor the apparent improved avidity of C-DDD-Fab-hMN-14 over the parentalIgG is that the Gly/Ser linkers in the former provide for a moreflexible molecule than IgG. Although the N-DDD variants also possessflexible peptide linkers, the CEA binding sites are positioned close toone another and adjacent to the DDD dimer, resulting in reduced avidity.

Example 15 Serum Stability of TF1 and TF2

TF1 and TF2 were designed to be stably tethered structures that could beused in vivo where extensive dilution in blood and tissues would occur.The stability of TF2 in human sera was assessed using BIACORE. TF2 wasdiluted to 0.1 mg/ml in fresh human serum, which was pooled from fourdonors, and incubated at 37° C. under 5% CO₂ for seven days. Dailysamples were diluted 1:25 and then analyzed by BIACORE using an IMP-239HSG sensorchip. An injection of WI2 IgG was used to quantify the amountof intact and fully active TF2. Serum samples were compared to controlsamples that were diluted directly from the stock. TF2 is highly stablein serum, retaining 98% of its bispecific binding activity after 7 days(not shown). Similar results were obtained for TF1 in either human ormouse serum (not shown).

Example 16 Biodistribution of TF2 in Tumor-Bearing Mice

The biodistribution studies were performed for TF2 in female athymicnude mice bearing s.c. human colorectal adenocarcinoma xenografts (LS174T). Cells were expanded in tissue culture until enough cells had beengrown to inject 50 mice s.c. with 1×10⁷ cells per mouse. After one week,tumors were measured and mice assigned to groups of 5 mice pertime-point. The mean tumor size at the start of this study was0.141±0.044 cm³. All the mice were injected with 40 μg ¹²⁵I-TF2 (250pmoles, 2 μCi). They were then sacrificed and necropsied at 0.5, 2, 4,16, 24, 48, and 72 hrs post-injection. A total of 35 mice were used inthis study. Tumor as well as various tissues were removed and placed ina γ-counter to determine percent-injected dose per gram (% ID/g) intissue at each time-point.

Radioiodination of ¹²⁵I-TF2 resulted in 2.7% unbound isotope with aspecific activity of 1.48 mCi/mg. The labeled sample was then subjectedto SE-HPLC alone and after mixing with a 20-fold molar excess of CEA.Approximately 83% of the TF2 eluted off with a retention time of 10.1minutes. There was 9% aggregated material (RT=9.03 min) and 8% lowmolecular weight material (RT=14.37 min) in the labeled TF2. When mixedwith CEA, 95% of the labeled TF2 shifted to a high molecular weighspecies (RT=7.25 min). These results indicated that the labeledpreparation was acceptable for administration to the tumor-bearing mice.

Table 1 presents the calculated % ID/g values in the tumors and varioustissues. Peak tumor uptake occurred at 4 h post-injection (10.3±2.1%ID/g). Between 16 and 24 h post-injection, the amount of TF2 in thetumor is not significantly different (5.3±1.1% ID/g and 5.37±0.7% ID/g),indicating that peptide could be administered anytime between these twotime-points, depending on blood values, without impacting tumortargeting. Uptake and clearance of TF2 from normal tissues was verysimilar to what has been observed previously for TF1. Both TF1 and TF2appeared to favor clearance through the RES system (spleen and liver).

The blood PK for TF2 in tumor-bearing mice were also evaluated and foundto exhibit biphasic clearance. These data were analyzed usingtwo-compartment analysis provided in the WinNonlin Nonlinear EstimationProgram (v. 4.1) and the parameters determined are shown in Table 2.

Example 17 Pretargeting with TF2 in Tumor Bearing Mice

A pretargeting study was performed with TF2 in female athymic nude micebearing s.c. human colorectal adenocarcinoma xenografts (LS 174T). Cellswere expanded in tissue culture until enough cells had been grown toinject 55 mice s.c. with 1×10⁷ cells per mouse. After one week, tumorswere measured and mice assigned to groups of 5 mice per time-point. Themean tumor size at the start of this study was 0.105±0.068 cm³. Twentymice were injected with 80 μg ¹²⁵I-TF2(500 pmoles, 2 μCi) and 16 h lateradministered ^(99m)Tc-IMP-245 (40 μCi, 92 ng, 50 pmoles). The mice weresacrificed and necropsied at 0.5, 1, 4, and 24 h post-peptide injection.In addition, 3 mice of the 24 h time-point groups were imaged on aγ-camera at 1, 4, and 24 h post-injection. As a control, 3 additionalmice received only ^(99m)Tc-IMP-245 (no pretargeting) and were imaged at1, 4, and 24 h post-injection, before being necropsied after the 24 himaging session. Tumor as well as various tissues were removed andplaced in a y-counter to determine % ID/g in tissue at each time-point.

The % ID/g values determined for ¹²⁵I-TF2 and ^(99m)Tc-IMP-245pretargeted with ¹²⁵I-TF2 are summarized in Tables 3 and 4,respectively. TF2 levels remained relatively unchanged over the first 4h following injection of the peptide (or 20 h post-TF2 administration),ranging from 6.7±1.6% ID/g at 0.5 h post-peptide injection (16.5 hpost-TF2 administration) to 6.5±1.5% ID/g at the 4 h time-point (20 hpost-TF2 injection). Tumor uptake values (% ID/g) of IMP-245 pretargetedwith TF2 were 22±3%, 30±14%, 25±4%, and 16±3% at 0.5, 1, 4, and 24 hpost-peptide injection.

In terms of normal tissues, there was significantly less peptide in theliver, lungs, and blood at each time-point examined in the micepretargeted with TF2 in comparison to the results obtained with otherpretargeting agents developed to date (Rossi, et al. Clin Cancer Res.2005; 11(19 Suppl): 7122s-7129s). These data indicate that the TF2clears efficiently through normal organs without leaving behind anyresidual fragments that might bind subsequently administered peptide.

The high tumor uptake coupled with lower levels in normal tissuesyielded excellent tumor:non-tumor (TINT) ratios (Table 5), thusvalidating TF2 as a suitable pretargeting agent for localizingdi-HSG-based effectors to CEA-producing tumors.

Example 18 Generation of TF2 Using a Glutathione Redox System

As an alternative embodiment to the methods disclosed in Examples 13 and14 above, a stably tethered structure such as TF1 or TF2 may begenerated using a glutathione redox system to form specific disulfidebonds linking the stably tethered structure together.

A simplified and efficient method for generating TF2 was accomplished asfollows. The entire process was conducted at room temperature.C-DDD2-Fab-hMN-14 (Protein L-purified) and h679-Fab-AD2(IMP-291-purified) were first mixed in roughly stoichiometricconcentrations in 1 mM EDTA, PBS, pH 7.4. Reduced glutathione was addedto a final concentration of 1 mM. After 30 minutes, oxidized glutathionewas added to a final concentration of 2 mM. BIACORE analysisdemonstrated that TF2 formation was 50% complete 2 minutes afteraddition of oxidized glutathione and 100% complete within 4 hours. TF2was purified to near homogeneity by IMP-291 affinity chromatography asdescribed in Example 14 above.

Example 19 Site-Specific Pegylation of Ganulocyte MacrophageColony-Stimulating Factor (GM-CSF)

Recombinant human GM-CSF (14 kDa) is used clinically to treat a varietyof hematological disorders. A limitation of current GM-SCF products isshort circulating half-lives, which therefore must be administered topatients by daily injection for optimal effectiveness. One approach thathas been used to prolong the circulation half-lives of proteintherapeutics is to modify the protein with polyethylene glycol (PEG) toincrease its effective size. However, all present methods known forconjugating PEG to proteins (pegylation) are not optimal, and usuallyrequire modification of the protein of interest to achieve site-specificcoupling (Doherty et al., Bioconjugate Chem. 2005, 16: 1291-1298). Evenwith such modifications, the conjugation yields are varied and theresulting products may not be homogenous.

Site-specific pegylation of GM-CSF with quantitatively yield can beachieved with the present invention (hereafter referred to as theDock-and-Lock (DNL) method or technology) as outlined below. The DDD2sequence is fused to the C-terminus of GM-CSF via a spacer to produce adimer of GM-SCF, creating a docking site for AD2, which is conjugated toPEG to obtain PEG-AD2. The formation of pegylated GM-CSF results bycombining GM-CSF-DDD2 and PEG-AD2 under similar conditions as describedfor TF2. It is noted that in addition to prolonging the circulationhalf-lives, the dimeric structure of GM-CSF in the pegylated productshould be more potent than the current monomeric form of GM-CSF. Thisstrategy can be applied to other cytokines (such as recombinant humanIL-2), enzymes (such as recombinant human arginase), or biologicallyactive peptides (such as the peptide agonist of the thrombopoietinreceptor, see Cwirta et al., Science 1997, 276: 1696-1699) or peptidemimetics that have a need for longer circulation half-lives to improvetherapeutic efficacy.

Example 20 Novel Immunodrugs Enabled by the DNL Technology

A fusion protein as a B component that will allow the conjugation of acytotoxic drug of interest can be produced and used for coupling to atargeting protein produced as an A component, resulting in a novel typeof immunodrug as outlined below. First, a well-expressed immunoglobulinhuman light chain is selected as the scafold or carrier protein, whichis fused to the AD2 sequence at its C-terminus. To prevent the formationof light chain dimer, the terminal cysteine (which forms a disulfidelinkage with the Fd chain) is replaced with a serine. Further, at leastone N-glycosylation site (the tripeptide sequence N-X-T) is engineeredinto the light chain to enable the addition of oligosaccharides, whichcan be produced recombinantly in high yield, purified to homogeneity,and used as a substrate for drug conjugation via appropriatechemistries, for example, as described by Shih et al for the conjugationof anthracyclin to amino-dextran (Cancer Res. 1991; 51: 4192-4198). Suchdrug-containing B-components can be combined with a variety of Acomponents comprising DDD2 linked to a binding structure that possessesthe targeting and internalization functions. Alternatively, adrug-containing amino-dextran derivatized with AD2 is combined with asuitable A component to enable target specific drug therapy. Otherwell-expressed recombinant molecules can also be selected as thescaffold or carrier proteins for drug conjugation.

Example 21 Targeting of Pathogens to Neutrophils for Kill

A broad-spectrum anti-infective agent potentially useful for treatingthe diseases caused by a variety of pathogens including influenza Avirus, Candida albicans, and E. coli has been reported recently for achemical conjugate comprising recombinant human surfactant proteinfragment D (rfhSP-D) and the Fab of an anti-CD89 antibody (Tacken etal., J. Immunol. 2004, 172: 4934-4940). The DNL technology can be usedfor producing stably tethered complexes that will also target pathogensto neutrophils for kill as follows. A truncated fragment of hSP-Dcomprising the α-helical coiled coil neck domain and the C-terminalcarbohydrate recognition domains (CRDs) is fused at the N-terminus toDDD2 to generate an A structure that binds multivalently to a pathogenthrough CRDs. To provide targeting for the FcRs on neutrophils the Astructure is linked to a B component composed of a fusion protein ofanti-CD89 Fab and AD2, resulting in a stable complex composed of twoCRDs of hSP-D and one Fab of anti-CD89. Similar anti-infective agentscan be prepared by substituting human surfactant protein A (hSP-A) forhSP-D and other antibodies such as those for CD3 and CD64.

Example 22 Multivalent, Multispecific Structures Generated withProtein-Protein Interaction Domains not Derived from PKA and AKAPs

Two basic strategies are envisioned. The first strategy depends onsearching and evaluating other naturally occurring protein-proteininteraction domains that may be suitable for substituting the roles ofDDD and AD. For example, the N-terminal dimerization domain of HNF-1amay replace DDD and the dimerization cofactor for HFN-1 (DcoH) mayreplace AD. The second strategy is outlined below.

Human p53 is a modular protein consisting of discrete functionaldomains. The C-terminal residues 325-355 (Scheme I) of human p53, termedthe tetramerization domain (p53tet), spontaneously form a tetramer insolution, which is in fact a dimer of dimers with a weak affinity (Kd ˜2uM) between the two dimers. However, the two monomers in each dimer arestrongly associated, with a Kd reported to be lower than 10⁻¹⁵ M (Brokxet al., J. Biol. Chem. 2003; 278: 2327-2332). Fusion proteins containingp53tet are therefore expected to form very tightly bound dimers, asfusion proteins containing the DDD sequence of human RIIα of PKA. Toligate a second structure to the dimer of p53tet, binding peptides forp53tet with Kd of 1 uM or lower and containing 15 to 50 residues areselected using the yeast 2-hybrid system or a suitable phage displaylibraries. The peptide with the highest affinity (i.e. the lowest valuefor Kd) is derivatized with cysteine if necessary and fused to a proteinof interest, which can be stably tethered to the dimer of p53tet.

Scheme I GEYFTLQIRGRERFEMFRELNEALELKDAQA (SEQ ID NO: 28)

Production and Use of Hexavalent IgG-Based DNL Structures (HIDS) Example23 Hexameric Constructs

The DNL technology described above for formation of a₂b complexes wasapplied to generate hexavalent IgG-based DNL structures (HIDS). Twotypes of modules, which were produced as recombinant fusion proteins,were combined to generate a variety of HIDS. Fab-DDD2 modules were asdescribed for use in generating Tri-Fab structures (Rossi et al. ProcNatl Acad Sci USA. 2006; 103(18): 6841-6, see Examples above). TheFab-DDD2 modules form stable homodimers that bind to AD2-containingmodules. To generate HIDS, two types of IgG-AD2 modules were created topair with the Fab-DDD2 modules: C-H-AD2-IgG and N-L-AD2-IgG.

C-H-AD2-IgG modules have an AD2 peptide fused to the carboxyl terminus(C) of the heavy (H) chain of IgG via a 9 amino acid residue peptidelinker (FIG. 9A). The DNA coding sequences for the linker peptide(GSGGGGSGG, SEQ ID NO:29) followed by the AD2 peptide(CGQIEYLAKQIVDNAIQQAGC, SEQ ID NO:4) are coupled to the 3′ end of theCH3 (heavy chain constant domain 3) coding sequence by standardrecombinant DNA methodologies, resulting in a contiguous open readingframe. When the heavy chain-AD2 polypeptide is co-expressed with a lightchain polypeptide, an IgG molecule is formed possessing two AD2 peptides(FIG. 9B), which can therefore bind two Fab-DDD2 dimers. The C-H-AD2-IgGmodule can be combined with any Fab-DDD2 module to generate a widevariety of hexavalent structures composed of an Fc fragment and six Fabfragments. If the C-H-AD2-IgG module and the Fab-DDD2 module are derivedfrom the same parental monoclonal antibody (MAb) the resulting HIDS ismonospecific with 6 binding arms to the same antigen (FIG. 10). If themodules are instead derived from two different MAbs then the resultingHIDS are bispecific, with two binding arms for the specificity of theC-H-AD2-IgG module and 4 binding arms for the specificity of theFab-DDD2 module (FIG. 11).

N-L-AD2-IgG is an alternative type of IgG-AD2 module in which an AD2peptide is fused to the amino terminus (N) of the light (L) chain of IgGvia a 13 amino acid residue peptide linker (FIG. 12A). The L chain canbe either Kappa (K) or Lambda (λ) and will also be represented as K inthe text or Figures with the same meaning. The DNA coding sequences forthe AD2 peptide (CGQIEYLAKQIVDNAIQQAGC, SEQ ID NO:4) followed by thelinker peptide (GGGGSGGGSGGG, SEQ ID NO:30) are coupled to the 5′ end ofthe coding sequence for the variable domain of the L chain (VL),resulting in a contiguous open reading frame. When the AD2-kappa chainpolypeptide is co-expressed with a heavy chain polypeptide, an IgGmolecule is formed possessing two AD2 peptides (FIG. 12B), which cantherefore bind two Fab-DDD2 dimers. The N-L-AD2-IgG module can becombined with any Fab-DDD2 module to generate a wide variety ofhexavalent structures composed of an Fc fragment and six Fab fragmentsarranged as shown in FIG. 13.

Example 24 Creation of C-H-AD2-IgG-pdHL2 Expression Vectors

The pdHL2 mammalian expression vector has been used to mediate theexpression of many recombinant IgGs. A plasmid shuttle vector wasproduced to facilitate the conversion of any IgG-pdHL2 vector into aC-H-AD2-IgG-pdHL2 vector. The gene for the Fc (CH2 and CH3 domains) wasamplified using the pdHL2 vector as a template and the oligonucleotidesFc BglII Left and Fc Bam-EcoRI Right as primers.

Fc BglII Left (SEQ ID NO: 31) 5′-AGATCTGGCGCACCTGAACTCCTG-3′Fc Bam-EcoRI Right (SEQ ID NO: 32)5′-GAATTCGGATCCTTTACCCGGAGACAGGGAGAG-3′

The amplimer was cloned in the pGemT PCR cloning vector. The Fc insertfragment was excised from pGemT with XbaI and BamHI restriction enzymesand ligated with AD2-pdHL2 vector that was prepared by digestion ofh679-Fab-AD2-pdHL2 with XbaI and BamHI, to generate the shuttle vectorFc-AD2-pdHL2 (FIG. 14A).

To convert any IgG-pdHL2 expression vector (FIG. 14B) to aC-H-AD2-IgG-pdHL2 expression vector (FIG. 14C), an 861 bp BsrGI/NdeIrestriction fragment is excised from the former and replaced with a 952bp BsrGI/NdeI restriction fragment excised from the Fc-AD2-pdHL2 vector.BsrGI cuts in the CH3 domain and NdeI cuts downstream (3′) of theexpression cassette.

Example 25 Production of C-H-AD2-hLL2 IgG

Epratuzumab, or hLL2 IgG, is a humanized anti-human CD22 MAb. Anexpression vector for C-H-AD2-hLL2 IgG was generated from hLL2IgG-pdHL2, as described in Example 24, and used to transfect Sp2/0myeloma cells by electroporation. Following transfection, the cells wereplated in 96-well plates and transgenic clones were selected in mediacontaining methotrexate. Clones were screened for C-H-AD2-hLL2 IgGproductivity by a sandwich ELISA using 96-well microtitre plates coatedwith an hLL2-specific anti-idiotype MAb and detection withperoxidase-conjugated anti-human IgG. Clones were expanded to rollerbottles for protein production and C-H-AD2-hLL2 IgG was purified fromthe spent culture media in a single step using Protein-A affinitychromatography. SE-HPLC analysis resolves two protein peaks (FIG. 15).The retention time of the slower eluted peak (8.63 min) is similar tohLL2 IgG. The retention time of the faster eluted peak (7.75 min) isconsistent with a ˜300 kDa protein. It was later determined that thispeak represents disulfide linked dimers of C-H-AD2-hLL2-IgG. This dimeris reduced to the monomeric form during the DNL reaction. SDS-PAGEanalysis demonstrated that the purified C-H-AD2-hLL2-IgG consists ofboth monomeric and disulfide-linked dimeric forms of the module (FIG.16). Protein bands representing these two forms are evident by SDS-PAGEunder non-reducing conditions, while under reducing conditions all ofthe forms are reduced to two bands representing the constituentpolypeptides (Heavy chain-AD2 and kappa chain). No other contaminatingbands were detected.

Example 26 Production of C-H-AD2-hA20 IgG

hA20 IgG is a humanized anti-human CD20 MAb. An expression vector forC—H-AD2-hA20 IgG was generated from hA20 IgG-pDHL2, as described inExample 24, and used to transfect Sp2/0 myeloma cells byelectroporation. Following transfection, the cells were plated in96-well plates and transgenic clones were selected in media containingmethotrexate. Clones were screened for C-H-AD2-hA20 IgG productivity bya sandwich ELISA using 96-well microtitre plates coated with anhA20-specific anti-idiotype MAb and detection with peroxidase-conjugatedanti-human IgG. Clones were expanded to roller bottles for proteinproduction and C-H-AD2-hA20 IgG was purified from the spent culturemedia in a single step using Protein-A affinity chromatography. SE-HPLCand SDS-PAGE analyses gave very similar results to those obtained forC-H-AD2-hLL2 IgG in Example 25.

Example 27 Production of N-L-AD2-hA20 IgG

A 197 bp DNA duplex comprising the coding sequence for the light chainleader peptide, AD2, a 13-residue peptide linker and the first fourresidues of hA20 Vk (all in frame) was generated as follows. Two 100-mersynthetic oligonucleotides, which overlap by 35 base-pairs, were madefully duplex by primer extension using Taq polymerase.

LP-AD2-L13 Top (SEQ ID NO: 33)CATCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCGACGGCTGTGGCCAGATCGAGTACCTGGCCA AGCAGATCLP-AD2-L13 Bottom (SEQ ID NO: 34)CCGCCAGACCCGCCACCTCCGGACCCTCCGCCGCCGCAGCCGGCCTGCTGGATGGCGTTGTCCACGATCTGCTTGGCCAGGTACTCGATCTG GCCACAGC

The sequence was amplified by PCR, which appended XbaI and PvuIIrestriction sites to the 5′ and 3′ ends, respectively. The amplimer wascloned into pGemT.

LP-Left XbaI (SEQ ID NO: 35) TCTAGACACAGGACCTCATCATGGGATGGAGCTGTAL13-VK Right PvuII (SEQ ID NO: 36) CAGCTGGATGTCACCTCCGCCAGACCCGCCACCTCC

The 197 bp XbaI/PvuII fragment was excised from pGemT and ligated withthe hA20 VK shuttle vector h2B8-Vk-pBR2, which was prepared by digestionwith XbaI and PvuII. The new shuttle vector is AD2-K-hA20-pBR2. A 536 bpXbaI/Bam HI restriction fragment was excised from AD2-K-hA20-pBR2 andligated with hA20-IgG-pDHL2 vector that was prepared by digestion withXbaI and Bam HI to generate the expression vectorN-L-AD2-hA20-IgG-pdHL2.

N-L-AD2-hA20-IgG-pdHL2 was used to transfect Sp2/0 myeloma cells byelectroporation. Following transfection, the cells were plated in96-well plates and transgenic clones were selected in media containingmethotrexate. Clones were screened for N-L-AD2-hA20 IgG productivity bya sandwich ELISA using 96-well microtitre plates coated with anhA20-specific anti-idiotype MAb and detection with peroxidase-conjugatedanti-human IgG. Clones were expanded to roller bottles for proteinproduction and N-L-AD2-hA20 IgG was purified from the spent culturemedia in a single step using Protein-A affinity chromatography.

Size exclusion HPLC showed that the majority of the N-L-AD2-hA20 IgG inthe prep is in a monomeric form with a retention time similar to IgG.Two additional peaks likely representing disulfide linked dimeric andtrimeric forms and each accounting for approximately 15% of the totalprotein were also observed (FIG. 17A). Mild reduction of the prep, as isused in the DNL reaction, results in the conversion of the dimeric andtrimeric forms to the monomeric form (FIG. 17B). Sketches of theputative structures for the three forms are provided in FIG. 18.

Example 28 Generation of Hex-hA20

The DNL method was used to create Hex-hA20, a monospecific anti-CD20HIDS, by combining C-H-AD2-hA20 IgG (see Example 26) with hA20-Fab-DDD2.The Hex-hA20 structure contains six anti-CD20 Fab fragments and an Fcfragment (FIG. 10).

Hex-hA20 was made in four steps.Step 1, Combination: A 210% molar equivalent of (hA20-Fab-DDD2)₂ wasmixed with C-H-AD2-hA20 IgG. This molar ratio was used because twoFab-DDD2 dimers are coupled to each C-H-AD2-hA20 IgG molecule and anadditional 10% excess of the former ensures that the coupling reactionis complete. The molecular weights of C-H-AD2-hA20 IgG and(hA20-Fab-DDD2)₂ are 168 kDa and 107 kDa, respectively. As an example,134 mg of hA20-Fab-DDD2 would be mixed with 100 mg of C-H-AD2-hA20 IgGto achieve a 210% molar equivalent of the former. The mixture istypically made in phosphate buffered saline, pH 7.4 (PBS) with 1 mMEDTA.Step 2, Mild Reduction: Reduced glutathione (GSH) was added to a finalconcentration of 1 mM and the solution is held at room temperature(16-25° C.) for 1-24 hours.Step 3, Mild Oxidation: Following reduction, oxidized glutathione (GSSH)was added directly to the reaction mixture to a final concentration of 2mM and the solution was held at room temperature for 1-24 hours.Step 4, Isolation of the DNL product: Following oxidation, the reactionmixture was loaded directly onto a Protein-A affinity chromatographycolumn. The column was washed with PBS and the Hex-hA20 was eluted with0.1 M Glycine, pH 2.5. Since excess hA20-Fab-DDD2 was used in thereaction, there was no unconjugated C-H-AD2-hA20 IgG, or incomplete DNLstructures containing only one (hA20-Fab-DDD2)₂ moiety. The unconjugatedexcess hA20-Fab-DDD2 does not bind to the affinity resin; therefore, theProtein A-purified material contains only the desirable product.

The calculated molecular weight from the deduced amino acid sequences ofthe constituent polypeptides is 386 kDa. Size exclusion HPLC analysisshowed a single protein peak with a retention time consistent with aprotein structure of 375-400 kDa (FIG. 19). SDS-PAGE analysis undernon-reducing conditions shows a cluster of high molecular weight bandsindicating a large covalent structure (FIG. 20A, lane 3). SDS-PAGE underreducing conditions (FIG. 20B, lane 3) shows the presence of only thethree expected polypeptide chains: the AD2-fused heavy chain (HC-AD2),the DDD2-fused Fd chain (Fd-DDD2), and the kappa chains.

Example 29 Generation of Hex-hLL2

The DNL method was used to create a monospecific anti-CD22 HIDS(Hex-hLL2) by combining C-H-AD2-hLL2 IgG (see Example 25) withhLL2-Fab-DDD2. The DNL reaction was accomplished as described forHex-hA20 in Example 28.

The calculated molecular weight from the deduced amino acid sequences ofthe constituent polypeptides is 386 kDa. Size exclusion HPLC analysisshowed a single protein peak with a retention time consistent with aprotein structure of 375-400 kDa (FIG. 21). SDS-PAGE analysis undernon-reducing conditions shows a cluster of high molecular weight bandsindicating a large covalent structure (FIG. 20A, lane 4). SDS-PAGE underreducing conditions (FIG. 20B, lane 4) shows the presence of only thethree expected polypeptide chains: HC-AD2, Fd-DDD2, and the kappa chain.

Example 30 Generation of DNL1 and DNL1C

The DNL method was used to create bispecific HIDS by combiningC-H-AD2-hLL2 IgG (see Example 25) with either hA20-Fab-DDD2 to obtainDNL1 or hMN-14-DDD2 to obtain DNL1C. DNL1 has four binding arms for CD20and two for CD22. As hMN-14 is a humanized MAb to carcinoembryonicantigen (CEA), DNL1C has four binding arms for CEA and two for CD22. TheDNL reactions were accomplished as described for Hex-hA20 in Example 28.

For both DNL1 and DNL1C, the calculated molecular weights from thededuced amino acid sequences of the constituent polypeptides are ˜386kDa. Size exclusion HPLC analysis showed a single protein peak with aretention time consistent with a protein structure of 375-400 kDa foreach structure (DNL1 in FIG. 22A and DNL1C in FIG. 22B). SDS-PAGEanalysis under non-reducing conditions shows a cluster of high molecularweight bands indicating a large covalent structure (FIG. 20A, lanes 1 &5). SDS-PAGE under reducing conditions (FIG. 20B, lanes 1 & 5) showsthat the large covalent structures are composed solely of the threeexpected polypeptides: HC-AD2, Fd-DDD2, and the kappa chain.

Example 31 Generation of DNL2 and DNL2C

The DNL method was used to create bispecific HIDS by combiningC-H-AD2-hA20 IgG (see Example 26) with either hLL2-Fab-DDD2 to obtainDNL2 or hMN-14-DDD2 to obtain DNL2C. DNL2 has four binding arms for CD22and two for CD20. DNL2C has four binding arms for CEA and two for CD20.The DNL reactions were accomplished as described for Hex-hA20 in Example28.

For both DNL2 and DNL2C, the calculated molecular weights from thededuced amino acid sequences of the constituent polypeptides are ˜386kDa. Size exclusion HPLC analysis showed a single protein peak with aretention time consistent with a protein structure of 375-400 kDa foreach structure (FIG. 23). SDS-PAGE analysis under non-reducingconditions shows a cluster of high molecular weight bands indicating alarge covalent structure (FIG. 20A, lanes 2 & 6). SDS-PAGE underreducing conditions (FIG. 20B, lanes 2 & 6) shows that the largecovalent structures are composed solely of the three expectedpolypeptides: HC-AD2, Fd-DDD2, and the kappa chain.

Example 32 Generation of K-Hex-hA20

The DNL method was used to create a monospecific anti-CD20 HIDS(K-Hex-hA20) by combining N-L-AD2-hA20 IgG (see Example 27) withhA20-Fab-DDD2. The DNL reaction was accomplished as described forHex-hA20 in Example 28.

The calculated molecular weight from the deduced amino acid sequences ofthe constituent polypeptides is 386 kDa. SDS-PAGE analysis undernon-reducing conditions shows a cluster of high molecular weight bandsindicating a large covalent structure (FIG. 24, lanes 2 & 3). SDS-PAGEunder reducing conditions (FIG. 24, lane 2R & 3R) shows that the largecovalent structure is composed solely of the four expected polypeptides:Fd-DDD2, H-chain, kappa chain, and AD2-kappa.

Example 33 Generation of DNL3

A bispecific HIDS was generated by combining N-L-AD2-hA20 IgG (seeExample 27) with hLL2-Fab-DDD2. The DNL reaction was accomplished asdescribed for Hex-hA20 in Example 28.

The calculated molecular weight from the deduced amino acid sequences ofthe constituent polypeptides is 386 kDa. Size exclusion HPLC analysisshowed a single protein peak with a retention time consistent with aprotein structure of 375-400 kDa (FIG. 25). SDS-PAGE analysis undernon-reducing conditions shows a cluster of high molecular weight bandsindicating a large covalent structure (FIG. 24, lane 1). SDS-PAGE underreducing conditions (FIG. 24, lane 1) shows that the large covalentstructure is composed solely of the four expected polypeptides: Fd-DDD2,H-chain, kappa chain, and AD2-kappa.

Example 34 In Vitro Characterization of HIDS

As shown in FIGS. 26 and 27, the HIDS generated as described in Examples28-33 retain the binding properties of their parental Fab/IgGs.Competitive ELISAs were used to investigate the binding avidities of thevarious HIDS using either a rat anti-idiotype MAb to hA20 (WR2) toassess the binding activity of the hA20 components or a ratanti-idiotype MAb to hLL2 (WN) to assess the binding activity of thehLL2 components. To assess hA20 binding, ELISA plates were coated withhA20 IgG and the HIDS were allowed to compete with the immobilized IgGfor WR2 binding. To assess hLL2 binding, plates were coated with hLL2IgG and the HIDS were allowed to compete with the immobilized IgG for WNbinding. The relative amount of anti-Id bound to the immobilized IgG wasdetected using peroxidase-conjugated anti-Rat IgG.

The relative CD20 binding avidities are shown in FIG. 26A. DNL2, whichhas two CD20 binding groups, showed a similar binding avidity to hA20IgG, which also has two CD20-binding arms. DNL1, which has fourCD20-binding groups, had a stronger (˜4-fold) relative avidity than DNL2or hA20 IgG. Hex-hA20, which has six CD20-binding groups, had an evenstronger (˜10-fold) relative avidity than hA20 IgG.

Similar findings are shown for CD22 binding in FIG. 26B. DNL1, which hastwo CD20 binding groups, showed a similar binding avidity to hLL2 IgG,which also has two CD22-binding arms. DNL2, which has four CD22-bindinggroups, had a stronger (>5-fold) relative avidity than DNL1 or hLL2 IgG.Hex-hLL2, which has six CD22-binding groups, had an even stronger(>10-fold) relative avidity than hLL2 IgG.

As both DNL2 and DNL3 contain two hA20 Fabs and four hLL2 Fabs, theyshow similar strength in binding to the same anti-id antibody (FIG. 27).

Some of the HIDS were shown to have potent anti-proliferative activityon lymphoma cell lines. DNL1, DNL2 and Hex-hA20 inhibited cell growth ofDaudi Burkitt Lymphoma cells in vitro (FIG. 28). Treatment of the cellswith 10 nM concentrations was substantially more effective for the HIDScompared to rituximab (FIG. 28A). Using a cell counting assay, thepotency of DNL1 and DNL2 was estimated to be more than 100-fold greaterthan that of rituximab, while the Hex-hA20 was shown to be even morepotent (FIG. 28B). This was confirmed with an MTS proliferation assay inwhich dose-response curves were generated for Daudi cells treated with arange of concentrations of the HIDS (FIG. 29). Compared to rituximab,the bispecific HIDS (DNL1 and DNL2) and Hex-hA20 were >100-foldand >10000-fold more potent, respectively.

Example 35 In Vivo Anti-Tumor Activity of HIDS

The HIDS were shown to have therapeutic efficacy in vivo using a humanBurkitt Lymphoma model in mice (FIG. 30). Low doses (12 μg) of DNL2 andHex-hA20 more than doubled the survival times of tumor bearing mice.Treatment with higher doses (60 μg) resulted in long-term survivors.

Example 36 Comparative Effects of HIDS and Parent IgG on Lymphoma CellLines

Dose-response curves for HIDS (DNL1, DNL2, Hex-hA20) versus a parent IgG(hA20 IgG) were compared for three different lymphoma cell lines (FIG.31), using an MTS proliferation assay. In Daudi lymphoma cells (FIG. 31,top panel), the bispecific structures DNL1 (not shown) and DNL2showed >100-fold more potent anti-proliferative activity and Hex-hA20showed >10,000-fold more potent activity than the parent hA20 IgG.Hex-hLL2 and the control structures (DNL1-C and DNL2-C) had very littleanti-proliferative activity in this assay (not shown).

In Raji lymphoma cells (FIG. 31, middle panel), Hex-hA20 displayedpotent anti-proliferative activity, but DNL2 showed only minimalactivity compared with hA20 IgG. In Ramos lymphoma cells (FIG. 31,bottom panel), both DNL2 and Hex-hA20 displayed potentanti-proliferative activity, compared with hA20 IgG. These results showthat the increased potency of HIDS relative to the parent IgGs is notlimited to particular cell lines, but rather is a general phenomenon forcells displaying the appropriate targets.

Example 37 Effects of Cross-Linking on Efficacy of HIDS and Parent IgGs

Cross-linking of anti-CD20 monoclonal antibodies has been shown toenhance their efficacy in vitro. FIG. 32 shows the effects ofcross-linking on the relative efficacies of HIDS versus parent IgG,using an MTS assay. As shown in FIG. 32, this effect was replicated inDaudi lymphoma cells treated with hA20 IgG cross-linked with goatanti-human IgG Fc-specific cross-linker, compared with non-cross-linkedhA20 IgG. However, no enhancement of anti-proliferative activity wasobserved with DNL2 or Hex-hA20 in the presence of cross-linker. Asdiscussed below, it is possible that the Fc portion of the HIDS becomesinaccessible when four additional Fab groups are tethered to itscarboxyl termini.

Example 38 Stability in Serum

FIG. 33 shows the stability of DNL1 and DNL2 in human serum, asdetermined using a bispecific ELISA assay. The protein structures wereincubated at 10 μg/ml in fresh pooled human sera at 37° C. and 5% CO₂for five days. For day 0 samples, aliquots were frozen in liquidnitrogen immediately after dilution in serum. ELISA plates were coatedwith an anti-Id to hA20 IgG and bispecific binding was detected with ananti-Id to hLL2 IgG. Both DNL1 and DNL2 were highly stable in serum andmaintained complete bispecific binding activity.

Example 39 CDC and ADCC Activity

In vivo, anti-CD20 monoclonal antibodies such as rituximab and hA20 canutilize complement-dependent cytotoxicity (CDC), antibody-dependentcellular cytotoxicity (ADCC) and signal transduction induced growthinhibition/apoptosis for tumor cell killing. The hexavalent DNLstructures (DNL1, DNL2, Hex-hA20) were tested for CDC activity usingDaudi cells in an in vitro assay. Surprisingly, none of the hexavalentstructures that bind CD20 exhibited CDC activity (FIG. 34). The parenthA20 IgG exhibited potent CDC activity (FIG. 34), while as expected thehLL2 antibody against CD22 showed no activity. The lack of effect ofDNL2 and Hex-hA20 was of interest, since they comprise hA20-IgG-Ad2,which showed similar positive CDC activity to hA20 IgG (FIG. 34).

DNL1 was assayed for ADCC activity using freshly isolated peripheralblood mononuclear cells (FIG. 35). Both rituximab and hA20 IgG showedpotent activity on Daudi cells (FIG. 35), while DNL1 did not exhibit anydetectable ADCC activity.

These data suggest that the Fc region may become inaccessible foreffector functions (CDC and ADCC) when four additional Fab groups aretethered to its carboxyl termini. Therefore, the hexavalent DNLstructures appear to rely only on signal transduction induced growthinhibition/apoptosis for in vivo anti-tumor activity.

TABLE 1 Tumor Uptake and Tissue Clearance of ¹²⁵I-TF2 in LS 174TTumor-Bearing Nude Mice % ID/g ± SD Tissue 0.5 h 2 h 4 h 16 h 24 h 48 h72 h LS 174T 4.43 ± 1.13 9.19 ± 1.18 10.33 ± 2.05  5.32 ± 1.09 5.37 ±0.72 1.69 ± 0.60 1.00 ± 0.13 Liver 11.71 ±2.22  8.39 ± 0.86 4.24 ± 0.110.32 ± 0.02 0.26 ± 0.03 0.15 ± 0.02 0.12 ± 0.01 Spleen 22.04 ± 6.02 24.87 ± 8.22  15.39 ± 1.35  0.73 ± 0.14 0.45 ± 0.06 0.25 ± 0.08 0.21 ±0.03 Kidney 13.45 ± 0.64  6.31 ± 0.48 3.88 ± 0.24 0.31 ± 0.04 0.24 ±0.03 0.14 ± 0.02 0.11 ± 0.01 Lungs 9.02 ± 1.38 4.99 ± 0.62 3.91 ± 0.080.33 ± 0.06 0.23 ± 0.04 0.09 ± 0.00 0.06 ± 0.01 Blood 36.17 ± 3.49 15.51 ± 2.43  9.06 ± 0.93 0.68 ± 0.07 0.43 ± 0.05 0.16 ± 0.04 0.11 ±0.03 Stomach 3.03 ± 0.45 26.00 ± 5.55  50.79 ± 10.83 0.85 ± 0.10 1.08 ±0.38 0.23 ± 0.06 0.20 ± 0.03 Sm. Int. 2.21 ± 0.17 3.09 ± 0.50 2.08 ±0.11 0.19 ± 0.03 0.18 ± 0.04 0.06 ± 0.01 0.05 ± 0.01 Lg. Int. 0.83 ±0.03 1.38 ± 0.12 1.62 ± 0.07 0.21 ± 0.04 0.25 ± 0.05 0.07 ± 0.01 0.09 ±0.03 Tail 3.83 ± 0.16 3.64 ± 0.95 2.79 ± 0.38 0.19 ± 0.02 0.18 ± 0.050.09 ± 0.02 0.06 ± 0.01 Tumor 0.154 ± 0.040 0.098 ± 0.055 0.114 ± 0.0610.175 ± 0.061 0.159 ± 0.014 0.240 ± .150  0.468 ± 0.220 wt (g)

TABLE 2 Blood pharmacokinetics of TF2 in LS174T tumor bearing micet_(1/2)α t_(1/2)β Cmax CL (h) (h) (ng) (ng/h * ng) 0.58 ± 0.08 3.47 ±0.36 31,186 ± 995 0.51 ± 0.02

TABLE 3 Tumor Uptake and Tissue Clearance of TF2 in LS 174TTumor-Bearing Nude Mice % ID/g ± SD Time post-TF2 16.5 h 17 h 20 h 40 hTime post-IMP245 0.5 h 1 h 4 h 24 h LS 174T 6.7 ± 1.6 9.0 ± 4.9 6.5 ±1.5 3.5 ± 0.8 Liver 0.29 ± 0.03 0.35 ± 0.05 0.27 ± 0.02 0.14 ± 0.02Spleen 0.49 ± 0.12 0.53 ± 0.10 0.46 ± 0.08 0.22 ± 0.06 Kidney 0.48 ±0.11 0.45 ± 0.14 0.29 ± 0.06 0.14 ± 0.01 Lungs 0.31 ± 0.04 0.37 ± 0.090.24 ± 0.06 0.12 ± 0.03 Blood 0.53 ± 0.05 0.61 ± 0.16 0.44 ± 0.11 0.20 ±0.04 Stomach 1.05 ± 0.13 1.78 ± 0.64 0.88 ± 0.47 0.50 ± 0.40 Sm. Int.0.20 ± 0.02 0.27 ± 0.09 0.13 ± 0.03 0.08 ± 0.03 Lg. Int. 0.30 ± 0.100.47 ± 0.17 0.20 ± 0.06 0.10 ± 0.05 Tail 0.41 ± 0.13 0.26 ± 0.06 0.22 ±0.15 0.09 ± 0.01 Tumor wt (g) 0.279 ± 0.142 0.222 ± 0.113 0.362 ± 0.2320.356 ± 0.152

TABLE 4 Tumor Uptake and Tissue Clearance of ^(99m)Tc-IMP-245pretargeted with TF2 in LS 174T Tumor-Bearing Nude Mice % ID/g ± SD Timepost-TF2 16.5 h 17 h 20 h 40 h Time post-IMP245 0.5 h 1 h 4 h 24 h LS174T 21.8 ± 3.0  30.1 ± 13.7 25.0 ± 3.7  16.3 ± 2.9  Liver 0.64 ± 0.070.41 ± 0.06 0.23 ± 0.06 0.14 ± 0.02 Spleen 0.59 ± 0.07 0.30 ± 0.06 0.16± 0.08 0.09 ± 0.02 Kidney 8.7 ± 1.4 5.0 ± 0.4 2.4 ± 0.4 1.2 ± 0.2 Lungs1.6 ± 0.2 0.69 ± 0.16 0.24 ± 0.05 0.10 ± 0.03 Blood 1.7 ± 0.2 0.50 ±0.12 0.11 ± 0.02 0.04 ± 0.01 Stomach 0.37 ± 0.09 0.87 ± 1.28 0.09 ± 0.080.16 ± 0.09 Sm. Int. 0.79 ± 0.04 1.08 ± 0.22 0.25 ± 0.12 0.15 ± 0.06 Lg.Int. 0.30 ± 0.09 0.13 ± 0.03 1.9 ± 2.0 0.40 ± 0.28 Tail 2.1 ± 0.4 0.94 ±0.45 0.45 ± 0.49 0.06 ± 0.02 Tumor wt (g) 0.279 ± 0.142 0.222 ± 0.1130.362 ± 0.232 0.356 ± 0.152

TABLE 5 T/NT ratio for the pretargeted ^(99m)Tc-peptide (IMP-245) usingTF2. Time post - IMP245 0.5 h 1 h 4 h 24 h Liver 34 ± 4  83 ± 10 116 ±32 115 ± 21 Spleen 37 ± 4 109 ± 21 170 ± 54 177 ± 30 Kidney   3 ± 0.4  7± 2 11 ± 2 14 ± 3 Lungs 14 ± 2 47 ± 4 106 ± 26 162 ± 24 Blood 13 ± 2 66± 5 237 ± 36 395 ± 26 Stomach  63 ± 25  169 ± 116  456 ± 271 135 ± 91Sm. Int. 28 ± 3 35 ± 5 114 ± 47 125 ± 46 Lg. Int.  75 ± 17 241 ± 31  22± 14  57 ± 34 Tail 11 ± 3 37 ± 8  164 ± 135 293 ± 80

TABLE 6 Examples of Complexes Comprised of Two Types of Antigen-BindingSubunits Target of A Target of B A B Application CEA HSG hMN-14 h679pRAIT; cancer imaging/therapy CEA In-DTPA hMN-14 h734 pRAIT/cancerimaging/therapy ED-B fibronectin HSG L19 h679 pRAIT/cancerimaging/therapy ED-B fibronectin In-DTPA L19 h734 pRAIT/cancerimaging/therapy CD20 CD22 hA20 hLL2 Lymphoma and autoimmune disease(AID) therapies CD22 CD20 hLL2 hA20 Lymphoma/AID therapies CD19 CD20Lymphoma/AID therapies EGFR IGFR1 Solid tumor therapy VEGFR1/Flt-1VEGFR2/KDR Blocking VEGF/PlGF binding; solid tumor and angiogenesistherapies VEGFR3/Flt-4 VEGFR2/KDR Blocking angiogenesis and solid tumortherapies CD19 CD3/TCR Lymphoma/AID therapies CD19 CD16/FcγRIIIaLymphoma/AID therapies CD19 CD64/FcγRI Lymphoma/AID therapies HER2/neuCD89/FcαRI Breast cancer therapy HER2/neu CD16 Breast cancer therapyHER2/neu CD64 Breast cancer therapy HER2/neu CD3 breast cancer therapyCD30 CD64 Lymphoma therapy CD33 CD64 Acute myeloid leukemia (AML)therapy EGFR CD2 Solid tumor therapy EGFR CD64 Solid tumor therapy EGFRCD16 Solid tumor therapy EGFR CD89 Solid tumor therapy PfMSP-1 CD3Malaria therapy HN CD3 HN1,4 c OKT3 Tumor vaccine enhancer HN CD28 HN1,4c 15E8 Tumor vaccine enhancer EpCAM/17-1A CD3 Solid tumor therapyIL-2R/Tac CD3 Lymphoma/AID therapies CA19-9 CD16 Solid tumor therapyMUC1 CD64 Solid tumor therapy HLA class II CD64 L243 Cancer therapyG_(D2) CD64 Neuroblastoma therapy G250 CD89 Renal cell carcinoma therapyTAG-72 CD89 hCC49 Solid tumor therapy EpCAM Adenovirus fiber knobRetargeting viral vector-solid tumor therapy PSMA Adenovirus fiber knobProstate cancer therapy CEA Adenovirus fiber knob S11 CEA-positivecancer therapy HMWMAA Adenovirus fiber knob Melanoma therapy G250Adenovirus fiber knob Renal cell carcinoma therapy CD40 Adenovirus fiberknob S11 Immune disease and cancer therapies M13 coat protein Alkalinephosphatase Viral detection GpIIb/IIIa tPA 7E3 P4B6 Enhancingthrombolysis

TABLE 7 Examples of Complexes Comprised of One Type of Antigen-Bindingand One Type of Effector Subunit Target of A A B Application CD74 hLL1Rap (N69Q) CD74+ Cancer/AID therapies CD22 hLL2 Rap (N69Q) Lymphoma andautoimmune disease (AID) therapies MUC1 hPAM4 Rap (N69Q) Pancreaticcancer therapy EGP-1 hRS7 Rap (N69Q) Solid cancer therapy IGF1R hR1 Rap(N69Q) Solid tumor therapy CD22 HLL2/RFB4 PE38 Lymphoma therapy CD30PE38 Lymphoma therapy CD25/Tac PE38 Lymphoma therapy Le^(Y) PE38 Solidtumor therapy Mesothelin PE38 Solid tumor therapy Erb-B2 PE38 Breastcancer EpCAM PE38 Solid tumor therapy CD25 dgA Lymphoma therapy CD30 dgALymphoma therapy CD19 dgA Lymphoma therapy CD22 dgA Lymphoma therapy CD3DT390 Graft-versus host disease CD25 PLC Lymphoma therapy Gp240 GeloninMelanoma therapy X Anti-X Straptavidin ELISA X Anti-X HRP ELISA X Anti-XAP ELISA X Anti-X GFP Reporter protein GpIIb/IIIa 7E3 tPA Enhancingthrombolysis X Anti-X Cytokine Retargeting a cytokine X Anti-X Growthfactor Retargeting a growth factor X Anti-X Soluble receptor componentRetargeting a receptor X Anti-X Carboxypeptidase G2 (CPG2) Prodrugtherapy X Anti-X penicillinamidase Prodrug therapy X Anti-X β-lactamaseProdrug therapy X Anti-X Cytosine deaminase Prodrug therapy X Anti-XNitroreductase Prodrug therapy p97 L49 E. coli beta-galactosidaseProdrug therapy X Anti-X Human carboxyesterase 2 Solid cancer therapy

TABLE 8 Examples of Complexes with Two Different Types of EffectorSubunits Target of A Target of B A B Application IL-4R — IL-4 PE38 Solidtumor therapy — IL-4R PE38 IL-4 Solid tumor therapy IL-4 IL-13 sIL-4RsIL-13R Asthma, allergy therapy IL-13 IL-4 sIL-13R sIL-4R Asthma,allergy therapy VEGFR-2 — VEGF₁₂₁ Shiga-like toxin Cancer therapyVEGFR-2 VEGF₁₂₁ Diptheria toxin Cancer therapy ED-B fibronectin ILGF-1Cancer therapy

TABLE 9 Examples of Complexes With One Type of Antigen-Binding SubunitTarget of A A Application X Anti-X Treating or detecting a diseasebearing the X marker CD14 Anti-CD14 Treating septic shock CD111/nectin-1Anti-CD111 Treating herpesvirus infection Folate receptor α Treatingfilovirus infection (e.g. Ebola and Marburg viruses) Gp120 TreatingHIV-1/AIDS IL-6 Treating myeloma, arthritis and other autoimmune diseaseIL-5 Treating asthma IL-8 Treating general infection CD154 Treatinglupus, transplant rejection, AID IgE Treating asthma LFA-1 Treatingtransplant rejection β-tryptase Treating allergy, inflammationCD105/endoglin Anti-angiogenesis GpIIb/IIa 7E3 Thrombolysis TNFα(Humira) AID therapy TNFα (remicade) AID therapy IgE (Xolair) Asthmatherapy RSV F-protein (Synagis) RSV therapy A1B1 of CEA hMN-15Inhibiting adhesion/invasion/metastasis of solid cancers N domain of CEAhMN-15 Inhibiting adhesion/invasion/metastasis of solid cancers

TABLE 10 Examples of Complexes with One Type of Effector Subunit Targetof A A Application Cytokine receptor Cytokine Enhancing cytokinefunction Growth factor receptor Growth Enhancing growth factor functionfactor Membrane bound Soluble Enhancing the capacity and avidityreceptor receptor of a soluble receptor component components Blood clottPA Enhancing the efficacy of tPA TPO receptor TPO Enhancing theefficacy of Thrombopoietin EPO receptor EPO Enhancing the efficacy ofrHuEPO TNFα sTNFα-R Enhancing the efficacy of Enbrel

1. A method of treating cancer comprising administering to a subjectwith cancer a hexavalent stably tethered structure comprising: (i) anIgG antibody that binds to a tumor-associated antigen (TAA); and (ii)four copies of an antibody fragment attached to the IgG antibody,wherein the antibody fragment binds to the same TAA or a different TAAas the IgG.
 2. The method of claim 1, wherein the antibody fragmentbinds to the same TAA as the IgG.
 3. The method of claim 1, wherein theantibody fragment binds to a different TAA from the IgG.
 4. The methodof claim 1, wherein the IgG comprises a first fusion protein and theantibody fragment comprises a second fusion protein and the first andsecond fusion proteins bind to each other.
 5. The method of claim 4,wherein the first fusion protein comprises an IgG antibody attached to afirst peptide, the second fusion protein comprises an antibody fragmentattached to a second peptide, and the first peptide binds to the secondpeptide.
 6. The method of claim 5, wherein the first peptide iscovalently attached to the second peptide by a disulfide bond.
 7. Themethod of claim 1, wherein the tumor-associated antigen is selected fromthe group consisting of carbonic anhydrase IX, alpha-fetoprotein,BrE3-antigen, CA125, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21,CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD138,colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, EGFR, EGP-1,EGP-2, Ep-CAM, Flt-1, Flt-3, folate receptor, G250 antigen, HLA-DR,human chorionic gonadotropin (HCG) and its subunits, HER2/neu, hypoxiainducible factor (HIF-1), Ia, IL-2, IL-6, IL-8, insulin-like growthfactor-1 (IGF-1), IGF-1 receptor (IGF-1R), KC4-antigen, KS-1-antigen,KS1-4, Le-Y, macrophage migration inhibitory factor (MIF), MAGE, MUC1,MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, antigen specific for PAM-4antibody, placental growth factor, p53, prostatic acid phosphatase, PSA,PSMA, RS5, S100, TAC, TAG-72, tenascin, TRAIL receptors, Tn antigen,Thomson-Friedenreich antigens, tumor necrosis factor, VEGF, ED-Bfibronectin, and 17-1A-antigen.
 8. The method of claim 1, wherein theIgG antibody and the antibody fragment are selected from the groupconsisting of LL1 (anti-CD74), LL2 (anti-CD22), RFB4 (anti-CD22), A20(anti-CD20), L243 (anti-HLA class II), CC49 (anti-TAG-72), MN-14(anti-CEA), MN-15 (anti-CEA), 679 (anti-HSG), 734 (anti-In-DTPA), L19(anti-ED-B fibronectin), R1 (anti-IGF-1R), PAM4 (anti-MUC1), RS7(anti-EGP-1), adalimumab, infliximab, omalizumab and palivizumab.
 9. Themethod of claim 1, wherein the IgG antibody and the antibody fragmentare chimeric, humanized or human.
 10. The method of claim 1, wherein theIgG antibody and the antibody fragment are not conjugated to anytherapeutic agent.
 11. The method of claim 10, further comprisingadministering at least one therapeutic agent to the subject.
 12. Themethod of claim 11, wherein the therapeutic agent is selected from thegroup consisting of a chemotherapeutic agent, a cytokine, a chemokine,an anti-angiogenic agent, an apoptotic agent, a drug, a prodrug, atoxin, an enzyme, a radioisotope, an immunomodulator, an antibiotic, anda hormone.
 13. The method of claim 1, wherein at least one therapeuticagent is attached to the hexavalent stably tethered structure.
 14. Themethod of claim 13, wherein the therapeutic agent is selected from thegroup consisting of a chemotherapeutic agent, a cytokine, a chemokine,an anti-angiogenic agent, an apoptotic agent, a drug, a prodrug, atoxin, an enzyme, a radioisotope, an immunomodulator, an antibiotic, anda hormone.
 15. The method of claim 1, wherein the cancer is selectedfrom the group consisting of acute lymphoblastic leukemia, acutemyelogenous leukemia, biliary cancer, breast cancer, cervical cancer,chronic lymphocytic leukemia, chronic myelogenous leukemia, colorectalcancer, endometrial cancer, esophageal, gastric, head and neck cancer,Hodgkin's lymphoma, lung cancer, medullary thyroid cancer, non-Hodgkin'slymphoma, multiple myeloma, renal cancer, ovarian cancer, pancreaticcancer, glioma, melanoma, liver cancer, prostate cancer, and urinarybladder cancer.
 16. The method of claim 15, wherein the cancer is aB-cell leukemia or B-cell lymphoma.
 17. The method of claim 1, furthercomprising administering a chemotherapeutic agent, a cytokine, radiationtherapy, immunotherapy, radioimmunotherapy, localized hyperthermia,laser irradiation, an anti-angiogenic agent or surgical excision incombination with the stably tethered structure.
 18. The method of claim1, wherein the stably tethered structure comprises a combination of IgGantibody and antibody fragment selected from the group consisting ofanti-CD74 X anti-CD20, anti-CD74×anti-CD22, anti-CD22×anti-CD20,anti-CD20×anti-HLA-DR, anti-CD19×anti-CD20, anti-CD20×anti-CD80,anti-CD2×anti-CD25, anti-CD8×anti-CD25, anti-CD2×anti-CD147,anti-CEACAM5×anti-CD3, anti-CEACAM6×anti-CD3, anti-EGFR×anti-CD3,anti-HER2/neu×anti-CD3, anti-CD20×anti-CD3, anti-CD74 X anti-CD3 andanti-CCD22×anti-CD3.
 19. The method of claim 12, wherein the therapeuticagent is selected from the group consisting of anthracyclines,epipodophyllotoxins, taxanes, antimetabolites, alkylating agents,antibiotics, COX-2 inhibitors, antimitotics, antiangiogenic agents,proapoptotic agents, doxorubicin, methotrexate, taxol, CPT-11,5-fluorouracil, bleomycin, busulfan, carboplatin, chlorambucil,cisplatin (CDDP), cyclophosphamide, dactinomycin, daunorubicin, estrogenreceptor binding agents, etoposide (VP16), farnesyl-protein transferaseinhibitors, gemcitabine, ifosfamide, mechlorethamine, melphalan,mitomycin, navelbine, nitrosourea, plicomycin, procarbazine, raloxifene,tamoxifen, taxol, temazolomide, transplatinum, vinblastine, vincristine,camptothecans, nitrogen mustards, alkyl sulfonates, nitrosoureas,triazenes, folic acid analogs, pyrimidine analogs, purine analogs,platinum coordination complexes and hormones.
 20. The method of claim12, wherein the radionuclide is selected from the group consisting of²¹²Pb, ²¹²Bi, ²¹³Bi, ²¹¹At, ²²³Ra, ²²⁵Ac, ³²P, ³³P, ⁴⁷Sc, ⁶⁷Cu, ⁶⁷Ga,⁸⁹Sr, ⁹⁰Y, ¹¹¹Ag, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁶⁶Dy, ¹⁷⁷Lu,¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹¹¹In, ¹⁹¹Os, ^(193m)Pt, ^(195m)Pt and ^(195m)Hg.21. The method of claim 12, wherein the anti-angiogenic agent isselected from the group consisting of angiostatin, baculostatin,canstatin, maspin, anti-VEGF antibodies, anti-P1GF peptides andantibodies, anti-vascular growth factor antibodies, anti-Flk-1antibodies, anti-Flt-1 antibodies and peptides, laminin peptides,fibronectin peptides, plasminogen activator inhibitors, tissuemetalloproteinase inhibitors, interleukin-12, IP-10, Gro-β,thrombospondin, 2-methoxyoestradiol, proliferin-related protein,carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate,angiopoietin-2, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin,paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin,AGM-1470, platelet factor 4 and minocycline.
 22. The method of claim 12,wherein the immunomodulator is selected from the group consisting ofcytokines, stem cell growth factors, lymphotoxins, hematopoieticfactors, interleukins, colony-stimulating factors, interferon-α,interferon-β, interferon-γ, S1 factor and TNF-alpha.
 23. The method ofclaim 22, wherein the cytokine is selected from the group consisting ofhuman growth hormone, N-methionyl human growth hormone, bovine growthhormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; follicle stimulating hormone (FSH), thyroid stimulatinghormone (TSH), luteinizing hormone (LH); hepatic growth factor;prostaglandin, fibroblast growth factor; prolactin; placental lactogen,OB protein; tumor necrosis factor-α, tumor necrosis factor-β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); NGF-β; platelet-growth factor; TGF-α, TGF-β;erythropoietin (EPO); macrophage-CSF (M-CSF); granulocyte-macrophage-CSF(GM-CSF); granulocyte-CSF (G-CSF); IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, IL-21, LIF, FLT-3, angiostatin, thrombospondin,endostatin, tumor necrosis factor and LT.
 24. The method of claim 5,wherein the first peptide has an amino acid sequence selected from thegroup consisting of SEQ ID NO:3 and SEQ ID NO:4 and the second peptidehas an amino acid sequence selected from the group consisting of SEQ IDNO:1 and SEQ ID NO:2.
 25. A method of killing cancer cells comprisingexposing the cancer cells to a hexavalent stably tethered structurecomprising: (i) an IgG antibody that binds to a tumor-associated antigen(TAA); and (ii) four copies of an antibody fragment attached to the IgGantibody, wherein the antibody fragment binds to the same TAA or adifferent TAA as the IgG.
 26. The method of claim 25, wherein theantibody fragment binds to the same TAA as the IgG.
 27. The method ofclaim 25, wherein the antibody fragment binds to a different TAA fromthe IgG.
 28. The method of claim 25, wherein the IgG comprises a firstfusion protein and the antibody fragment comprises a second fusionprotein and the first and second fusion proteins bind to each other. 29.The method of claim 28, wherein the first fusion protein comprises anIgG antibody attached to a first peptide, the second fusion proteincomprises an antibody fragment attached to a second peptide, and thefirst peptide binds to the second peptide.
 30. The method of claim 29,wherein the first peptide is covalently attached to the second peptideby a disulfide bond.
 31. The method of claim 25, wherein thetumor-associated antigen is selected from the group consisting ofcarbonic anhydrase IX, alpha-fetoprotein, BrE3-antigen, CA125, CD1,CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30,CD33, CD38, CD45, CD74, CD79a, CD80, CD138, colon-specific antigen-p(CSAp), CEA (CEACAM5), CEACAM6, EGFR, EGP-1, EGP-2, Ep-CAM, Flt-1,Flt-3, folate receptor, G250 antigen, HLA-DR, human chorionicgonadotropin (HCG) and its subunits, HER2/neu, hypoxia inducible factor(HIF-1), Ia, IL-2, IL-6, IL-8, insulin-like growth factor-1 (IGF-1),IGF-1 receptor (IGF-1R), KC4-antigen, KS-1-antigen, KS1-4, Le-Y,macrophage migration inhibitory factor (MIF), MAGE, MUC1, MUC2, MUC3,MUC4, NCA66, NCA95, NCA90, antigen specific for PAM-4 antibody,placental growth factor, p53, prostatic acid phosphatase, PSA, PSMA,RS5, 5100, TAC, TAG-72, tenascin, TRAIL receptors, Tn antigen,Thomson-Friedenreich antigens, tumor necrosis factor, VEGF, ED-Bfibronectin, and 17-1A-antigen.
 32. The method of claim 25, wherein theIgG antibody and the antibody fragment are selected from the groupconsisting of LL1 (anti-CD74), LL2 (anti-CD22), RFB4 (anti-CD22), A20(anti-CD20), L243 (anti-HLA class II), CC49 (anti-TAG-72), MN-14(anti-CEA), MN-15 (anti-CEA), 679 (anti-HSG), 734 (anti-In-DTPA), L19(anti-ED-B fibronectin), R1 (anti-IGF-1R), PAM4 (anti-MUC1), RS7(anti-EGP-1), adalimumab, infliximab, omalizumab and palivizumab. 33.The method of claim 25, wherein the stably tethered construct is moreeffective to kill cancer cells than the parent IgG antibody.